Flexible materials can adapt their structures easily in response to external stimuli. For this reason, they are often used as sensors or actuators; they can show useful and unusual mechanical behaviour such as negative thermal expansion or negative compressibility. In a conceptually related manner, disordered materials navigate a shallow configurational landscape of degenerate states. This degeneracy also often heightens their susceptibility to external perturbations. This lecture will explore the fundamental design principles associated with structural flexibility and correlated disorder, and their role in a range of functional materials. Case studies taken from our own work will includie metal–organic frameworks, oxide ceramics, and frustrated magnets.

Quantum information theory is developing at a rapid pace and one can easily envisage a bright future, demonstrated by the growing number of quantum computing simulations offered at large scale computation facilities and by the construction of the first prototypes of quantum computers.

In this area, the contribution of crystallography, and especially of quantum crystallography, is seamlessly vital, because the inner mechanism at hearth in the transmission of signal is tightly connected with the chemical bonding, the electron charge and spin density distribution, and, ultimately, the wavefunction.

Quantum Crystallography [1,2,3] deals, in fact, with the application of quantum theory in crystallography. Among the various quantities investigated within this field, the electron delocalization and the spin electron density play a fundamental role in spintronic materials.

Over the years, several theoretical analysis emerged that can be applied to computed as well as to experimental electron densities. Moreover, the modelling techniques enable the reconstruction of many quantities from ever more precise and sophisticated experiments.

In this lecture, novel spintronic materials, such as magnetic coordination polymers [4,5], will be discussed, within the framework of recent quantum crystallographic studies. The role of the linkers in the magnetic exchange is not fully clear yet. A combinatorial approach, including studies on materials at high pressure shed light on the subtleties of exchange mechanisms.

References

[1] A. Genoni et al. Chemistry, Eur. J., 2018, 24, 10881-10905.

[2] P. Macchi Cryst. Rev. 2020, 26, 209-268.

[3] P. Macchi Quantum Crystallography: Fundamentals and applications 2022, De Gruyter to be published.

[4] Kubus, M., Lanza, A., Scatena, R., Dos Santos, LHR., Wehinger, B., Casati, N., Fioka, C., Keller, L., Macchi, P., Rüegg, C., Krämer, KW. (2018) Inorg. Chem. 57, 4934-4943.

[5] R. Scatena, R. D. Johnson, P. Manuel, P. Macchi, J. Mater. Chem. C 2020, 8, 12840–12847.

The advances over the last decade in ultrabright X-ray sources have reinvigorated interest in time-resolved structural biology. Importantly, many of the time-scales of interest to structural biology are in fact accessible using serial crystallographic approaches at synchrotrons. Therefore, although the experiments themselves remain challenging, a number of new beamlines and endstations are coming online at synchrotron sources to serve this growing community. This has been complemented by increasing exchange between synchrotron and XFEL researchers, sharing methods, data processing tools and even supporting experiments that make use of both types of light source.

To help structural biologists take full advantage of these new resources, considerable effort is being put in to help interested researchers to optimise their sample in terms of crystal quantity, size and quality, as well as determine how to trigger the reaction of interest as uniformly as possible in each crystal. Key here is that optimising the sample and ensuring that sufficient data are collected to provide clear electron density maps at each time point requires both regular beamtime access and rapid feedback during the experiment.

The T-REXX endstation on beamline P14 at PETRA III has been built to address these challenges by a collaborative team from EMBL, Universität Hamburg and the Max Planck Institute for the Structure and Dynamics of Matter. It is dedicated to serial crystallographic data collection, and has an open design that can accommodate a range of serial sample mounts as well as different reaction initiation methods. A number of tools have been developed by the T-REXX collaboration for sample preparation and mounting, as well as protocols for sample and data collection optimisation. Rapid feedback on hit rate and resolution is presented in the controls GUI, and automatic processing pipelines deliver first maps a few hours after data collection is complete.

In this presentation I will present the current state of T-REXX, including recent results, and some of the tools we have developed to facilitate sample optimsation and mounting, reaction initiation and data processing. I will also take a look at the wider field and highlight some of the remaining challenges and opportunities.

We are investigating bacterial and eukaryotic ribosomes and their functional complexes to obtain insights into the process of protein synthesis. Building on our studies aimed at revealing the structures of eukaryotic cytosolic and mitochondrial ribosomes, we are now investigating eukaryotic translation initiation, targeting of proteins to membranes, regulation of protein synthesis, and how viruses reprogram host translation. Previously, we studied how Hepatitis C virus genomic RNA can bind mammalian ribosomes to achieve translation of viral mRNAs in the absence of some canonical cellular translation initiation factors. With our recent research activities we contributed to the understanding of how SARS-CoV-2, the virus that is responsible for the COVID-19 pandemic, shuts off host translation to prevent cellular defence mechanisms against the virus (Schubert et al. 2020). Furthermore, using a combination of cryo-electron microsocpy and biochemical assays we also investigated the mechanism of programmed ribosomal frameshifting, one of the key events during translation of the SARS-CoV-2 RNA genome that leads to synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins (Bhat et al. 2021).

Schubert K, Karousis ED, Jomaa A, Scaiola A, Echeverria B, Gurzeler LA, Leibundgut M, Thiel V, Mühlemann O, Ban N. (2020) SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nat Struct Mol Biol. (10):959-966

Bhatt PR, Scaiola A, Loughran G, Leibundgut M, Kratzel A, McMillan A, O’ Connor KM, Bode JW, Thiel V, Atkins JF and Ban N, 2021, Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome, Science, doi: 10.1126/science.abf3546.

Single Particle Analysis (SPA) application of cryo-electron microscopy (cryo-EM) has become one of the dominating methods for 3D structure determination of a wide variety of biological macromolecules to understand their function, mechanism of action^[1] and protein ligand/drug interactions. However, as the popularity of this technique increases, so does the need for accessibility and improved efficiency. In this abstract, we describe two cryo-Transmission Electron Microscopes (cryo-TEMs), that are equivalent to home source X-ray diffractometers, but for cryo-EM.

The first is the Thermo Scientific Tundra cryo-TEM operating at 100kV with a semi-automated grid loading system and automated data collection for SPA. Tundra allows users to load the sample in an effortless and robust way. Using this new microscope, we solved structures of several soluble and membrane protein samples. Standard sample such as apoferritin protein (equivalent to lysozyme crystals for X-ray crystallography) was solved to 2.6 Å resolution. More challenging samples such as homo-pentameric human GABA_A (gamma-aminobutyric acid type A) receptor was resolved to 3.4 Å reconstruction. The GABA_A receptor is a small membrane protein and ligand-gated chloride-ion channel that mediates inhibitory neurotransmission. GABA_A receptors are important therapeutic drug targets and hence it is vital to understand the molecular mechanism by which these receptors mediate neurotransmission. After decades of efforts, in 2014, this same sample of GABA_A receptor was crystallized and structure resolved to 3.0 Å^[2]. With cryo-EM on Tundra, we obtained similar resolution without the need of crystallization and in near native conditions.

To further push for more automation and high-throughput, we used the Thermo Scientific Glacios^TM cryo-TEM. Glacios has an Autoloader^TM, with a robotic arm which can load 12 grids simultaneously and switch the grids automatically. To push for higher resolution, Glacios is also equipped with direct electron detector (DED) and can be combined with Selectris energy filter. Using this system, we achieved a 2.4 Å resolution cryo-EM map for the same GABA_A receptor. Both these microscopes are not only good for sample screening and optimization but are also capable for generating high resolution structures comparable to those obtained from X-ray crystallography experiments.

Flaviviruses pose a complex threat to human health including a few global pathogens and numerous viruses with an epidemic potential. In the context of the co-circulation of closely-related viruses, non-neutralising immune responses may aggravate subsequent heterologous infections. Sub-optimal responses to vaccination entails a similar risk. To address these challenges, a detailed structural understanding of flavivirus infectious particles is essential to characterise quaternary epitopes responsible for broadly protective responses or, on the contrary, deleterious immune responses. Immature-like features and conformational “breathing” in circulating virions have been linked to the latter prompting for a better understanding of structural transitions underpinning viral maturation.

Taking advantage of an insect-specific flavivirus (ISF), we have determined high-resolution structures of immature and mature particles revealing key features in the maturation process. First, we produced chimeric viruses between the ISF and medically-relevant flaviviruses. We show that the outer shell of the chimeric viruses is native, which allowed cryo-EM structure determination at high-resolution for West Nile virus, Murray Valley Encephalitis virus and dengue virus. The structure of the dengue virus chimera at a resolution of 2.5Å reveals lipid-like ligands with a structural role likely to be conserved across all pathogenic flaviviruses. The structure of the immature ISF particle at a resolution of 3.9Å shows how the stem region of the E protein, where these ligands bind, is remodelled during maturation. Unexpectedly, the immature spike adopts a topology where prM forms a central pillar rather than the peripheral drawstring proposed earlier (Fig. 1A). This topology implies a revised organisation of the immature virion, which supports a collapse model for viral maturation (Fig. 1B). In this model, folding down of prM onto the membrane guides the collapse of the trimeric spikes.

Together, these structures provide new avenues to target the stem regions of E and prM for the development of improved vaccines and new therapeutics. More generally, we propose that the chimeric platform could be a largely applicable tool to investigate flavivirus biology.

Most of the rhinoviruses, which are the leading cause of common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. Before genome release, rhinoviruses convert to activated particles that contain pores in the capsid, lack capsid proteins VP4, and have altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here we present the structures of the native rhinovirus 14 and rhinovirus14-ICAM-1 complex at a resolution of 2.6 and 2.4 Å. The structures revealed a mechanism by which binding of rhinovirus 14 to ICAM-1 primes the virus for activation and subsequent genome release. The attachment of rhinovirus 14 to ICAM-1 induces conformational changes in the virion, which include translocation of the C-termini of VP4 subunits towards twofold symmetry axes of the capsid. Thus, VP4 subunits become poised for release through pores that open in the capsid upon particle activation. The cryo-EM reconstruction of rhinovirus 14 virion contains the resolved density of octa-nucleotides from the RNA genome, which interact with VP2 subunits near two-fold symmetry axes of the capsid. VP4 subunits with altered conformation, induced by the binding of rhinovirus 14 to ICAM-1, block the RNA-VP2 interactions and expose patches of positively charged residues around threefold symmetry axes of the capsid. The conformational changes of the capsid induce reorganization of the virus genome. The rearrangements of the capsid and genome probably lower the energy barrier of conversion of rhinovirus 14 virions to activated particles. The structure of rhinovirus 14 in complex with ICAM-1 represents an essential intermediate in the pathway of enterovirus genome release.

Enolase, a conserved glycolytic enzyme, that catalyzes the conversion of 2-phosphoglycerate (2PG) to phosphoenol pyruvate, important for energy production is an essential enzyme for mycobacterial growth. However, additionally enolase is a known moonlighting protein with additional functions in the cytoplasm as well as on the cell surface. It plays an important role in Mtb virulence by acting as cell surface receptor of human plasminogen. To derive a mechanistic insight into the function of this enzyme, we have deciphered the atomic level details of Mtb enolase structure in native as well as 2PG/PEP bound forms by both XRD and CryoEM microscopy. Notably, through X-ray structure superimposition of the enolase/2PG bound structures shows two binding confirmations of the 2PG in the active site. The cryoEM structure reveals the octameric conformation of Mtb enolase. P-P docking and simulation studies of enolase and plasminogen helps us to understand the molecular interaction of the complex.

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan component of the extracellular matrix (ECM), which facilitates important structural and signalling interactions between cells and their surroundings. The principal enzyme responsible for extracellular HS breakdown is heparanase (HPSE), an endo-glucuronidase of the CAZy GH79 family. Whilst normal HPSE activity is essential for HS processing, excessive HPSE overexpression weakens HS networks in the ECM, leading to increased cell mobility and release of growth factors stored by HS. Thus HPSE is an oncogene whose overexpression promotes metastasis in a range of cancers.

In this talk, I will give an overview of our work in this area over the last few years, covering our initial structural investigations into the molecualr basis of HPSE activity, the development of probes to visualize HPSE in tissues, and most recently, the structure guided rational design of HPSE inhibitors as anti-metastatic agents.

References

L. Wu, C. M. Viola et al (2015), Nat. Struct. Mol. Biol. (22) 1016–1022

L. Wu, J. Jiang, Y. Jin et al (2017), Nat. Chem. Biol. (13) 867–873

Fatty Acid Photodecarboxylase (FAP) is a recently discovered photoenzyme that catalyzes the conversion of fatty acids into alkane and CO₂ under light, with potential importance in green chemistry applications [1]. Its mechanism was still not fully understood and partly relied on a low-resolution crystal structure obtained from crystals with a twinning default [1]. Here, we present high-resolution crystal structures of FAP obtained in the dark and after light illumination at cryogenic temperatures (Figure 1). Combined with structural, computational, and spectroscopic techniques we are now able to provide a detailed reaction cycle of FAP. The reaction mechanism starts with an electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved CO₂ unexpectedly transforms in 100 ns, most likely into bicarbonate. This is orders of magnitude faster than in solution, which indicates a catalytic step. FT-IR, structural and functional studies on variants centered on two conserved active site residues (R451 and C432) showed that R451 is essential for substrate stabilization and proton transfer. Altogether this study provides a detailed characterization of this unique enzyme and reveals a striking and unanticipated mechanistic complexity [2].

[1] Sorigué D, Légeret B, Cuiné S, Blangy S, Moulin S, Billon E, Richaud P, Brugière S, Couté Y, Nurizzo D, Müller P, Brettel K, Pignol D, Arnoux P, Li-Beisson Y, Peltier G, Beisson F. (2017) Science. 357, 903.

[2] Sorigué, D., K. Hadjidemetriou, S. Blangy, G. Gotthard, A. Bonvalet, N. Coquelle, P. Samire, A. Aleksandrov, L. Antonucci, A. Benachir, S. Boutet, M. Byrdin, M. Cammarata, S. Carbajo, S. Cuiné, R. B. Doak, L. Foucar, A. Gorel, M. Grünbein, E. Hartmann, R. Hienerwadel, M. Hilpert, M. Kloos, T. J. Lane, B. Légeret, P. Legrand, Y. Li-Beisson, S. L. Y. Moulin, D. Nurizzo, G. Peltier, G. Schirò, R. L. Shoeman, M. Sliwa, X. Solinas, B. Zhuang, T. R. M. Barends, J.-P. Colletier, M. Joffre, A. Royant, C. Berthomieu, M. Weik, T. Domratcheva, K. Brettel, M. H. Vos, I. Schlichting, P. Arnoux, P. Müller, F. Beisson (2021) Science 372, 148.

Lytic polysaccharide monooxygenases (LPMOs) have been intensely studied since their first characterization in 2010 as a unique class of copper enzymes capable of oxidizing carbohydrates. LPMOs require the input of electrons and of O₂ or H₂O₂ to achieve hydroxylation of one carbon in the glycosidic bond. We focus on three aspects of the LPMO’s reaction mechanism: 1) What are the structural determinants of O₂ and H₂O₂ binding? 2) How do conserved second shell residues contribute to activity? 3) Does the O₂ based mechanism follow a superoxyl, hydroperoxyl or oxyl catalytic pathway? The ability to pinpoint hydrogen atoms to determine protonation states at and around the active site through the catalytic pathway is key to decipher the chemistry catalyzed by LPMOs. To achieve this, we combine high resolution X-ray and neutron protein crystallography to deliver precise, all atom structures of key reaction intermediates that can reveal i) the positions and interactions of all hydrogen atoms in the enzyme, ii) atomistic details of the active site without perturbing the metal oxidation state, and iii) the chemical nature of the activated dioxygen species coordinated to the active site copper. We will present our recent X-ray and neutron crystallographic studies that provide new insights into the LPMO mechanism.

The discovery of novel enzymes from antibiotic production pathways is nowadays a topic of utmost importance due to worldwide concerns with the increased resistance of pathogenic bacteria to antibiotics. In this work, we used a combination of X-ray crystallography, SAXS, and biochemical studies to identify the molecular fingerprints for a novel glucosamine kinase (GlcNK) family potentially implicated in antibiotic biosynthesis in Actinobacteria. We determined the high-resolution structure of a bacterial GlcNK in apo form and in complex with its biological substrates, providing unparalleled structural evidence of a transition state of the phosphoryl-transfer mechanism in this unique family of enzymes (PDB IDs 6HWJ, 6HWK and 6HWL; Fig. 1a-c). Conservation of glucosamine-contacting residues across a large number of uncharacterized proteins unveiled a specific glucosamine binding sequence motif. As result, a new UniProt annotation rule was created (MF_02218; Fig. 1d). The structural characterization of this enzyme provides new insights into the role of these unique GlcNKs as the missing link for the incorporation of environmental glucosamine to the metabolism of important intermediates in antibiotic production [1].

[1] Manso, J. A., Nunes-Costa, D., Macedo-Ribeiro, S., Empadinhas, N., Pereira, P. J. B. (2019). mBio. 10, e00239-19.

Mycobacteria are priority pathogens in terms of drug resistance worldwide and efforts aimed at deciphering their unique metabolic pathways and unveiling new targets for innovative drugs should be intensified. In particular, nontuberculous mycobacteria (NTM) are environmental organisms increasingly associated to opportunistic infections [1] and known to produce methylmannose polysaccharides (MMP). MMP have been implicated in the metabolism of precursors of cell envelope lipids crucial for stress resistance and pathogenesis. Although the functions of MMP remain to be confirmed experimentally, their tight interactions with fatty acids are intrinsically associated to unique and extensive methylation patterns, resulting from the action of hitherto uncharacterized methyltransferases.

In this work, we identified and characterized biochemically a novel mycobacterial methyltransferase (MeT1) that specifically blocks the non-reducing end of a MMP precursor. We crystallized and determined the first X-ray structure of the SAM-dependent MeT1 from M. hassiacum in complex with magnesium and its exhausted cofactor, SAH. In particular, the three high-resolution 3D structures (in space groups P3₂21 and C222₁; PDB entries 6H40, 6G7D and 6G80) in combination with SAXS data (SASBDB entry SASDDJ6) unveiled a dimeric arrangement of the enzyme in solution and a highly flexible lid important for its catalytic cycle. This structural information, together with molecular docking simulations, allowed the elucidation of the enzyme’s reaction mechanism, furthering our knowledge of MMP biosynthesis and providing important tools to dissect the role of MMP in NTM physiology and resilience [2].

[1] Falkinham III, J. O., (2015). Clin. Chest. Med. 36, 35.

[2] Ripoll-Rozada, J., Costa, M., Manso, J. A., Maranha, A., Miranda, V., Sequeira, A., Rita Ventura, M,. Macedo-Ribeiro, S., Pereira, P. J. B., Empadinhas, N. (2019). Proc. Natl. Acad. Sci. U.S.A. 116, 835.

We thank SOLEIL, ESRF and ALBA for provision of synchrotron radiation facilities, and their staff for help with data collection. This work was funded in part by national funds through Fundação para a Ciência e a Tecnologia (Portugal) through PhD Fellowship SFRH/BD/101191/2014 (to M.C.); the European Social Fund through Programa Operacional Capital Humano in the form of Postdoctoral Fellowship SFRH/BPD/108004/2015 (to J.R.-R.); the European Regional Development Fund (FEDER), through Centro2020 Project CENTRO-01-0145- FEDER-000012-HealthyAging2020 in the form of a postdoctoral fellowship (to A.M.); and the COMPETE 2020–Operational Programme for Competitiveness and Internationalization (POCI), PORTUGAL 2020 in the form of projects POCI-01-0145-FEDER-029221 (PTDC/BTM-TEC/29221/2017), “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), UID/NEU/04539/2013, and Research Unit MOSTMICRO (UID/CQB/04612/2013).

Time-resolved serial femtosecond crystallography (TR-SFX) at X-ray free electron lasers (XFELs) allows studying the structural dynamics of crystalline biological macromolecules down to the sub-picosecond time scale [1]. According to a pump-probe scheme, optical pump pulses initiate activity in light sensitive crystalline proteins and XFEL pulses generate diffraction patterns that allow determining intermediate-state structures. We apply TR-SFX to study light-induced dynamics in a reversibly photoswitchable fluorescent protein, rsEGFP2.

Reversibly photoswitchable fluorescent proteins are essential tools in advanced fluorescence nanoscopy of live cells. They can be repeatedly toggled back and forth between a fluorescent (on) and a non-fluorescent (off) state by irradiation with light at two different wavelengths. Our consortium (*) combines TR-SFX at XFELs, ultrafast absorption spectroscopy and simulation methods to study photoswitching intermediates in rsEGFP2 on the picosecond to nanosecond time scale. We have been able to identify the transient structure of rsEGFP2 in its excited state 1 ps after photoexcitation, and to observe the chromophore in a twisted state, midway between the stable configurations of the on and off states [2]. This observation, together with a ground-state intermediate structure determined 10 ns after photoexcitation, has allowed us to uncover details of the photo-switching mechanism of rsEGFP2 [3].

Based on the reaction intermediates determined by TR-SFX [2, 3] two rationally designed mutants of the rsEGFP2 have been generated. Pico- to nanosecond TR-SFX results experiments on these rsEGFP2 variants have been carried out at SACLA and the LCLS and provide insight into modified energy landscapes (unpublished).

[1] Colletier, J-P., Schirò, G. & Weik, M. (2018). Time-Resolved Serial Femtosecond Crystallography, Towards Molecular Movies of Biomolecules in Action in X-ray Free Electron Lasers: A Revolution in Structural Biology, edited by Fromme, P., Boutet, S., Hunter. M. Eds., Springer International Publishing, 11:331-356[2] Coquelle. N., Sliwa. M., Woodhouse. J., Schiro. G., Adam. A. … Colletier, J-P., I. Schlichting. & M. Weik. (2018). Nat. Chem. 10, 31-37 [3] Woodhouse. J., … Sliwa. M., Colletier, J-P., I. Schlichting. & M. Weik. (2020). Nat. Comm. 11, 1-11

Keywords: x-ray free electron lasers; time-resolved studies; photoswitchable fluorescent proteins

(*) the work presented involves a consortium composed of researchers from the Institut de Biologie Structurale, Grenoble, France, Institut Laue Langevin, Grenoble, France, Max-Planck-Institut for Medical Research, Heidelberg, Germany, RIKEN SPring-8 Center, Sayo, Japan, Linac Coherent Light Source, Menlo Park, USA, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, Laboratoire de Spectrochimie Infrarouge et Raman, Lille, France, Department of Physics, University of Rennes, France, Laboratoire de Chimie-Physique, CNRS/University Paris-Sud, University Paris-Saclay, Orsay, France, namely, Adam V., Andreeva E., Aquila A., Banneville A-S., Barends T., Bourgeois D., Boutet S., Byrdin M., Cammarata M., Carbajo S., Colletier J-P., Coquelle N., Demachy I., Doak B., Feliks M., Field M., Fieschi F., Foucar L., Gorel A., Grünbein M., Guillon V., Hilpert M., Hunter M., Jakobs S., Joti Y., Kloos M., Koglin J., Lane T., Liang M., Levy B., de la Mora E., Nass-Kovacs G., Owada S., Richard J., Robinson J., Roome. C., Ruckebusch C., Schirò G., Schlichting I., Seaberg M., Shoeman R., Sierra R., Sliwa M., Stricker M., Tetreau G., Thepaut M., Tono K., Uriarte L., Woodhouse J., Yabashi M., You D., Zala N. and Weik M.

In this talk, I will describe our recent work on the design and preparation of novel supramolecular architectures based on a range of element-based non-covalent interactions such as halogen bonds, chalcogen bonds, and pnictogen bonds. In addition to standard wet chemistry and slow evaporation methods, the utility of mechanochemical and cosublimation techniques will be discussed. Considered together, these methods enable a broad exploration of the polymorphic cocrystalline landscape. For example, the cosublimation approach overcomes an anticooperative halogen-bonding effect to produce fully saturated cocrystals of the tritopic halogen bond donor 1,3,5-trifluoro-2,4,6-triiodobenzene with 1,4-diazabicyclo[2.2.2]octane.¹ I also report on dicyanoselenodiazole and dicyanotelluradiazole derivatives which work as promising supramolecular synthons with the ability to form double chalcogen bonds with a wide range of electron donors including halides and oxygen‐ and nitrogen‐containing heterocycles.² In addition to X-ray diffraction, solid-state multinuclear magnetic resonance (SSNMR) and nuclear quadrupolar resonance (NQR) spectroscopies are employed to characterize all products and to establish spectral signatures for the various classes of bonds. Given the elements involved in these bonds, we report on a wide range of nuclides including e.g., ¹⁷O, ³¹P, ^35/37Cl, ⁷⁷Se, ^79/81Br, ¹²⁵Te, ¹²⁷I, ^121/123Sb, etc. As most of these are quadrupolar nuclides, the utility of specialized NMR techniques and very high applied magnetic fields will be discussed. In favourable cases, in-situ solid-state NMR spectroscopy allows for real-time monitoring of cocrystallization reactions and for the determination of activation energies.³

1. Szell, P. M. J.; Gabriel, S. A.; Caron-Poulin, E.; Jeannin, O.; Fourmigué, M.; Bryce, D. L. Cryst. Growth Des. 2018, 18, 6227. https://doi.org/10.1021/acs.cgd.8b01089

2. Kumar, V.; Xu, Y.; Bryce, D. L. Chem. Eur. J., in press. https://doi.org/10.1002/chem.201904795

3. Xu, Y.; Champion, L.; Gabidullin, B.; Bryce, D. L. Chem. Commun. 2017, 53, 9930.
https://doi.org/10.1039/C7CC05051H

Different crystallization and screening techniques have been developed for the discovery of new multicomponent molecular crystals. Exploring the polymorphic space for a given organic molecule typically includes searches across well-defined conditions, among others solvents, additives, and temperature. In recent years, especially mechanochemistry has been used intensively for the screening for new solid forms and as a promising, alternative method for accessing new polymorphs of active pharmaceutical ingredients (APIs) and API-cocrystals.[1-2] The ever-increasing interest in this method is contrasted by a still limited mechanistic understanding of the mechanochemical reactivity and selectivity. Furthermore, the influence of liquids used during the grinding on the polymorphic outcome is still far from being understood. Time-resolved in situ investigations of milling reactions provide direct insights in the underlying mechanisms.[3-5] We recently introduced different setups enabling in situ investigation of mechanochemical reactions using synchrotron XRD combined with Raman spectroscopy and thermography allowing to detect crystalline, amorphous, eutectic, and liquid intermediates. In this contribution, we will discuss our recent results investigating the formation of (polymorphic) cocrystals and salts, thereby elucidating the influence of solvents and seeds on the polymorph formation.[6-8] Our results indicate that in situ investigation of milling reactions offer a new approach to tune and optimize mechanochemical processes.

A careful investigation of the packing in the nearly 1500 organic, well-refined (R£0.050), P1, Z>1 structures archived in the 2019 version of the Cambridge Structural Database [1] has revealed that the molecules (or ions) in ca. 85% of those structures are related by obvious approximate symmetry that is periodic in at least two dimensions. An example is shown in Fig. 1. The nearly 250 P1, Z=1 structures of molecules that could lie on special positions were also analyzed; ca. 70% were found to have approximate symmetry.

Figure 1. Views of LUSMAN, P1, Z=2 [2]; the second view, of a bilayer, is rotated by 90° around the horizontal from the first. The approximate c211 symmetry of a layer (001) with 0.5 < z < 1.5 (axes [110], [10]; angle 89.9°) is obvious. The angles of those axes with c are 77.8° and 81.8°.

In only 8% of the Z>1 structures does it seem likely that refinement in a higher symmetry space group or smaller unit cell would have been preferable. That percentage is, however, much higher (39%) for P1 crystals of achiral or racemic material, which account for 11% of all Z>1 structures considered. For P1, Z>1 crystals that are enantiomerically pure the frequency of overlooked symmetry is only 2%. For the Z=1 crystals the percentage is 10% overall and 17% for the crystals of achiral or racemic material.

In the abstract of R. E. Marsh’s (1999) paper titled “P1 or P? Or something else?” [3] he wrote
In approximately one-third of the structures in which chiral molecules crystallize in P1 with Z=2, the two molecules are related by
an approximate center of inversion.
The present study found that 32% of the P1, Z=2 structures of enantiomerically pure material are P mimics. Molecular features that promote P mimicry have been identified; they may have implications for the probability of formation of solid solutions.

The approximate symmetry is often subperiodic, as it is in the example shown in Fig. 1. The ratio of structures having 2-D to those having 3-D approximate periodic symmetry is about 2:3 but the ratio is imprecise because of the difficulty of deciding on the dimensionality. In some structures the approximate symmetry is clearly 3-D and in others it is clearly 2-D, but in many others the dimensionality is at the 3-D/2-D borderline. In only 22 structures, however, was the approximate symmetry identified as 1-D. The approximate subperiodic symmetry was described with the labels for layer and rod groups found in Vol. E of International Tables [4].

The surprisingly exact approximate symmetry found in many P1 crystals could result from a distortion during growth or cooling of a more symmetric nucleus, but in more than 3% of the Z>1 structures quite different layers alternate so that the P1 symmetry must have been established at the time of crystal nucleation.

[1] Groom, C. R., Bruno, I. J., Lightfoot, M. P. & Ward, S. C. (2016), Acta Cryst. B72, 171-179. [2] Adam, W. & Zhang. A. (2003) Eur. J. Org. Chem. 2003, 587-591. [3] Marsh, R. E. (1999). Acta Cryst. B55, 931-936. [4] Kopský, V. & Litvin, D. B. (2002). Editors. International Tables for Crystallography, Vol. E, Subperiodic groups, Kluwer Academic Publishers, Dordrecht/Boston/London, 2002.

A 47-year-old polymorphic structure (Form δ) of the drug indomethacin (IDM) has been solved by 3D electron diffraction (3D ED). Since its discovery in 1974, the structure of δ-IDM had remained a mystery. By performing a unique crystallisation technique, we successfully cultivated its single crystal. The very thin, ribbon-like crystal was too small (~1 µm in width) to be studied by X-ray diffraction—including even the third generation of synchrotron radiation. With the aid of 3D ED, we finally elucidated the crystal structure of δ-IDM. The structure exhibits a very long b-axis with the slowest growth and shortest crystal dimension occurring along this direction. Consequently, reflections along 0k0 were missing in the 3D ED data and the structure could not be solved by direct methods. Instead, simulated annealing was employed to overcome this problem. This work highlights the powerfulness of 3D ED for structure determination of small crystals, which complements X-ray diffraction.

Non-Photochemical Laser-Induced nucleation (NPLIN) is a promising nucleation technique [1] for which more than eighty papers have been published. In an NPLIN experiment, a supersaturated solution of a molecule is irradiated by a laser (pulsed or continuous, focused or non-focused) that induces the molecule's nucleation. Even though glycine nucleation constitutes almost one-quarter of these research activities reported in the literature, the impact of different experimental conditions on its nucleation is still not fully understood [2]. NPLIN of glycine in water has been demonstrated at different molarities and different energy densities induced using a non-focalized pulsed laser (532 nm) at 290 K. A new index (Ind50), allowing easy comparison with the literature, was used to characterize the impact of molarities and energy densities on the nucleation efficiency. A threshold index (Ind_Thrs(β)) indicating the minimum energy density required to obtain in a given experimental condition one crystal per vials in average has been determined. The impact of the circular or linear polarization of the laser beam on the glycine polymorphism (α- or γ-glycine) has been studied and characterized using a third new index named NPLIN determinant. The experimental interface (glass-solution or air-solution) gives the opposite polymorphism behavior. The relationship between devices, solution, and experimental conditions and observable such as nucleation efficiency, nucleation site, induction time, crystal counting, and polymorphism have been modelized in a mind-map (Figure 1). Within this context, this work is a contribution towards a better understanding of the impact of experimental conditions on NPLIN nucleation that will permit a better design and control of NPLIN experimental setups.

[1] Garetz, B. A.; Aber, J. E.; Goddard, N. L.; Young, R. G.; Myerson, A. S. (1996) Phys. Rev. Lett. 77, 3475−3476.

[2] Clair, B.; Ikni, A.; Li, W.; Scouflaire, P.; Quemener, V.; Spasojević-de Biré, A. (2014) J. Appl. Crystallogr., 47, 1252−1260

Photo-induced electron transfer (PET) reactions are crucial for many biological and chemical reactions that occur in nature. Several studies are performed on many donor-bridge-acceptor (D-B-A) systems to have a better understanding of PET in terms of the rate of transfer and the overall geometry.[1] A mono-substituted pyrene derivative, pyrene-(CH₂)₂-N,N'-dimethylaniline, were designed where dimethylaniline (DMA) (electron donor) is connected to pyrene (electron acceptor) through alkane chain. Two polymorphic crystal forms, A and B, were crystallized in two separate crystallization batches in ethanol/ethyl acetate binary mixture. While, in the crystal structure A, pyrene and dimethylaniline are in axial orientation (P-1) with respect to each other, in B they are equatorial (P2₁/n). Studies on intramolecular PET has revealed the importance of conformational parameters of the molecules such as rotation around bonds that affects the distance and relative orientation of the donor and acceptor.[2] We have performed time-resolved (TR) pump-probe pink Laue X-ray diffraction experiments with the polymorphic crystals in ns time domain. TR pump-probe data was processed by RATIO[3] method by employing LaueUtil software[4]. The photodifference maps obtained from TR pump-probe diffraction measurements with polymorphic crystals, suggest electron transfer from DMA moiety. A thorough crystallographic and spectroscopic investigation with the polymorphic crystals, have allowed us to understand the important aspects of PET in this particular (D-B-A) system.

The in-surface crystallography of the family of three-periodic minimal surfaces (TPMS) consisting of the Primitive, Diamond, and Gyroid surfaces has been explored in detail in recent decades [1, 2]. We begin by reviewing the underlying group theory and geometry as well as the relationship between the TPMS and their universal covering space, hyperbolic two-space, the fundamentals of which are shown in Figure 1(a).

We describe how these methods can be used to tailor the topology and geometry of three-periodic nets realised as embeddings commensurate with the symmetries of these surfaces [3-5] as shown in Figure 1(b). Using these ideas, we demonstrate how a number of nets with pre-specified topological properties are readily produced in this manner and assess their relevance for further study in the context of reticular chemistry and soft matter materials science.

Finally, we present preliminary results on visualisation methods for understanding how these patterns and nets are realised in simulations of liquid crystals in bulk [6] and confined polymer systems. Using an array of methods from computational geometry, we visualise these simulations in hyperbolic two-space to facilitate easy comparisons and further analysis as shown in Figure 1(c).

The authors thank Anders Dahl, Myfanwy Evans, Olaf-Delgado Friedrichs, Benedikt Kolbe, Stuart Ramsden, Vanessa Robins, Gerd Schröder-Turk, and Monique Teillaud for discussions and feedback on these topics and results.

[1] Sadoc, J.-F. & Charvolin, J. (1989). Acta Crystallogr. A 45, 10-20.
[2] Robins, V., Ramsden, S., & Hyde, S. T. (2004). Eur. Phys. J. B 39(3), 365-375.
[3] Pedersen, M. C. & Hyde, S. T. (2018). Proc. Natl. Acad. Sci. U. S. A. 115(27), 6905-6910.
[4] Pedersen, M. C., Delgado-Friedrichs, O., & Hyde, S. T. (2018). Acta Crystallogr. A 74(3), 223-232.
[5] Hyde, S. T. & Pedersen, M. C. (2021). Proc. Roy. Soc. A 477, 20200372.
[6] Kirkensgaard, J. J. K., Evans, M. E., de Campo, L., & Hyde, S. T. (2014). Proc. Natl. Acad. Sci. U. S. A. 111(4), 1271-1276.

We introduce flat-branched semisimplicial (FBS) complexes as a universal language to describe aperiodic structures of finite local complexity. An FBS-complex naturally represents the set of local atomic arrangements occurring in the structure. It includes both metric and combinatorial data; the flexibility of the latter allows for incorporation of structural constraints on a longer range. An FBS-complex can embody "local rules" of any kind, whether or not they impose a perfect long-range order. We propose an algorithm for exploration of local rules in terms of an FBS-complex directly from the phased diffraction data. The FBS complex describing a structure entirely determines the density of atomic species, and yields experimentally verifiable constraints on their contribution to the structure factors.

The study of chromatic symmetries to describe physical properties of crystals and mapping of individual orientations in twins has extended the field of mathematical crystallography. This paper discusses chromatic, partially chromatic or achromatic properties of symmetries or partial operations of a coordinated coloring of a symmetrical pattern, which has a potential to describe a crystalline structure. Here, we consider a symmetrical pattern P consisting of disjoint congruent copies of a symmetrical motif M.

A coordinated coloring of P is a coloring that is perfect and transitive under the global symmetry group G of P, satisfying the condition that the coloring of M is also perfect and transitive under its symmetry group K (a local symmetry group). This means that G and K consists of elements that effect a permutation of the colors of the coloring of P and M respectively. If the coloring of P or M has two (respectively more than two) colors, then G or K is called a dichromatic (respectively polychromatic) symmetry group.

A global symmetry, a local symmetry, or a partial operation (an isometry of the plane that sends one motif to another, which may not be a global or local symmetry of P) of a coordinated coloring of P can be classified as either achromatic if it fixes all the colors; chromatic if it moves all the colors, and partially chromatic if it exchanges some colors, and fixes the rest.

As an example, consider the coordinated 4 – coloring of P given in the given figure. Each region in every motif is assigned a color from the set S={blue,yellow,red,green}. Every element of G=<2 0,0;m_[10];m_[01];z(1,0)>≅p2mm (a polychromatic symmetry group) permutes the colors in the 4 – coloring of P. We also note that in this coloring, every element of K=<4⁺ 0,0;m_[10]>≅4mm permutes the colors assigned to the triangles in the motif M. The dichromatic group of M is 4'mm': we have the 90^o rotation and two reflections that change colors, while two reflections fix colors. The dichromatic group of each of the other motifs is also 4'mm'.

The local chromatic symmetries of M exchanging blue and red are 4'⁺,4',m'_[11], and m'_{[11 ̅ ]}. On the other hand, the local achromatic symmetries of M fixing the colors blue and red are 2,m_[10] , and m_[01] . The global symmetries in P can be characterized as: z⁽⁴⁾ (1,0) (chromatic) sending blue to yellow, yellow to red, red to green, and green to blue; 2^(2,2) 0,0, and m^(2,2)_[01] (partially chromatic) exchanging yellow and green, and fixing red and blue; and m_[10] (achromatic) fixing all the colors. The 4-coloring of the frieze pattern is described by the group [p⁽⁴⁾ 2^(2,2) m^(2,2) m]⁽⁴⁾. The chromatic partial operations that map M to zM are also shown in the figure. Sending blue to yellow as well as red to green are z⁽²⁾ (1,0),2⁽²⁾ 1/2,0 ,m⁽²⁾_[01] 1/2,0 , and g⁽²⁾_[01] (1,0). Sending blue to green and red to yellow are 4⁽²⁾⁺ 1/2,1/2, 4^(2)- 1/2,(1/2) ̅, g⁽²⁾_[11](1/2,1/2) 1/2,0 and g⁽²⁾_{[11 ̅ ]}(1/2,1/2) 1/2,0.

In this talk, examples of coordinated colorings of Frieze and Plane Crystallographic patterns will be presented.

Superspace-group symmetry is essential to the unambiguous description of modulated structures, and a correct understanding of their physical properties. An exhaustive enumeration of superspace groups in up to 3+3 dimensions were announced in 2014. We now announce an exhaustive enumeration of magnetic superspace groups in up to 3+3 dimensions (over 250,000 groups). With these tables in hand, we have developed an algorithm and tool that detects the superspace-group (magnetic or non-magnetic) of an arbitrary modulated structure, given the amplitudes and phases of its modulations in P1 symmetry, and identifies it in the exhaustive symmetry-group table. This capability has been integrated into both the FINSYM and ISOCIF packages of the ISOTROPY software suite, and has been integrated with JANA 2000. The ISODISTORT package, which use group-representations to generate incommensurate structure models based on a given parent structure, now automatically identifies the unique magnetic superspace-group of each magnetically modulated child structure. Anyone can access these data sources and tools online to generate, symmetrize, transform, or otherwise explore magnetic or non-magnetic modulated structure models.

We present a classification of magnetic point groups which give an answer to the question: Which magnetic groups can describe a given magnetic mode?
There are 32 categories of magnetic point groups which describe 64 unique different magnetic modes: 16 with a ferromagnetic component and 48 without. This classification focused on magnetic modes is helpful for finding the magnetic space group which can describe the magnetic symmetry of the material.

The classification selects the magnetic space groups and the magnetic site-symmetry point groups which are compatible with a number of magnetic phenomena e.g. collinear antiferromagnetism and ferromagnetism, spin roerientation, antiferromagnetism with weak ferromagnetism. The use of our classification is demonstrated on a number of well-studied materials, e.g. alpha-Fe2O3, rare earth orthoferrites, RFeO3. It is particularly useful for materials with weak ferromagnetism. Examples of use for neutron powder diffraction studies are discussed in the context of the paper by Shirane (1958).

This presentation based on the paper (ib5097) recently accepted to Acta Cryst A.

X-ray diffraction from powders and single crystals has for decades been the key analytical tool in materials science. Bragg intensities provide information about the average crystals structure, but often it is disorder and specific local structure that control key material properties. For 1D data there has been an immense growth in combined analysis of Bragg and diffuse scattering using the Pair Distribution Function (PDF), and in our group we frequently use 1D PDF analysis to study nanocrystal nucleation in solvothermal processes [1] or thin films [2], or to analyse materials under operating conditions [3]. For single crystals, diffuse scattering studies have a long history with elaborate analysis in reciprocal space, but direct space analysis of the 3D-PDF is still in its infancy. We have used 3D-PDF analysis to study the crystal structures of high performance thermoelectric materials Cu₂Se (Fig 1) [4], PbTe [5], and 19-e half-heusler Nb_1-xCoSb [6], where the true local structure is essential for understanding the unique properties. For frustrated magnetic materials direct space analysis of diffuse magnetic scattering provides a new route to magnetic structures [7].

[1] N. L. N. Broge et al., Auto-catalytic formation of high entropy alloy nanoparticles, Angew. Chem. Intl. Ed., 59, 21920-21924 (2020)

[2] M. Roelsgaard et al., Time-Resolved Surface Pair Distribution Functions during Deposition by RF Magnetron Sputtering, IUCrJ, 6, 299–304 (2019)

[3] L. R. Jørgensen et al., Operando X-ray scattering study of thermoelectric β-Zn₄Sb₃, IUCr-J, 7, 100-104 (2020)

[4] N. Roth et al., Solving the disordered structure of β-Cu_2-xSe using the three-dimensional difference pair distribution function, Acta Crystallogr. Sect. A, 75, 465–473 (2019)

[5] K. A. U. Holm et al., Temperature Dependence of Dynamic Dipole Formation in PbTe, Phys. Rev. B, 102, 024112 (2020)

[6] N. Roth et al., A simple model for vacancy order and disorder in defective half-Heusler systems, IUCrJ, 7, 673-680 (2020)

[7] N. Roth et al., Model-free reconstruction of magnetic correlations in frustrated magnets, IUCr-J, 5, 410–416 (2018)

Many advanced functional properties of crystalline materials derive from complex disorder and short-range correlations that emerge from a subtle balance among competing interactions involving spin, charge, orbital, and strain degrees of freedom. Materials that harbor such disorder generally exhibit strongly enhanced responses, with electronic, magnetic, optical, and thermal properties that are extremely sensitive to perturbations such as magnetic or electric fields and are of considerable importance for future applications. Obtaining a detailed understanding of such complex disorder is required to control and exploit these unusual patterns that persist within short-range ordered states in order to access functional responses inaccessible to conventional, long-range ordered materials. Diffuse scattering is a powerful probe of such complex disorder and when measured from single crystals over large 3D volumes of reciprocal space provides detailed information regarding the existence and morphology of local distortions, as well as defect–defect correlations, i.e., the tendency for defects to cluster into nanoscale ordered structures [1,2].

Recent developments in instrumental advances now efficient measurements of single crystal diffraction data over large volumes of reciprocal space using synchrotron x-rays or neutrons. For the latter, dedicated instrumentation, in particular the Corelli instrument at the Spallation Neutron Source, has been constructed that enables measurements of such volumes with elastic discrimination [3]. The value of combining the complementarity of neutrons and x-rays of such measurements over large space of temperature and compositions will be demonstrated on recent investigation of relaxor ferroelectrics that provide new insight on the relation of local order to material properties relaxors [4]. While analyzing diffuse scattering data and obtaining detailed models of the underlying remains challenging, the availability of comprehensive measurements of the scattering over large 3D volumes enables new ways of analyzing the data, by utilizing the 3D-ΔPDF method [5]. This method allows to derive for example, direct, model free reconstructions of ionic correlations [6], which are essential in many energy materials, as well as magnetic correlations in frustrated magnets [7]. Recent advances in Machine Learning methods further provide invaluable and new, rapid insight into the information contained in these large data sets, in particular when measured over varying experimental parameters such as temperature or external fields [8].

[1] Welberry,T.R. Weber, T. (2016) Crystallography Reviews 22, 2-78[2] see contributions in Issue Diffuse Scattering (2005). Z. Kristallogr. Cryst. Mater. 220, Issue 12[3] Ye, F., et al. (2018). J. Appl. Cryst.. 51, 315 - 322.[4] Krogstad, M.J., et al. (2018). Nat. Mater. 17, 718 - 724.[5] Weber, T., Simonov, A. (2012). Z. Kristallogr. 225, 238.[6] Krogstad, M.J, et al. (2020). Nat. Mater. 19, 63 - 68.[7] Roth, N., May, A.F., Ye, F., Chakoumakos, B.C., Iversen, B.B. (2018). IUrJ. 5, 410.[8] Venderley, J., et al. (2020). Cond-Mat. Archiv arXiv:2008.03275.

Keywords: IUCr2020; abstracts; total scattering; single crystal diffuse scattering, complex disorder, short range correlations

Work supported by U.S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Science and Engineering Division

Many important functional materials are complex mixtures that derive their properties from the interplay of various individual component phases. In each case, the interfaces between phases are a crucial component in their own right, since they are the point at which much of the key chemistry (and/or physics) takes place [1, 2]. By their very nature, interfaces are notoriously more difficult to characterise than the bulk phases they connect; and the process of translating experimental measurements into a picture of atomic-scale structure remains a significant general challenge [3]. Here we explore the possibility that pair distribution function (PDF) measurements offer sensitivity to interface structure in a way that is strongly complementary to existing experimental and computational approaches.

Using a non-negative matrix factorisation (NMF) approach [4, 5], we show how the PDF of complex mixtures can be deconvolved into the contributions from the individual phase components and also the interface between phases. Our focus is on the model system Fe||Fe₃O₄. First, we establish proof-of-concept using idealised PDF data generated from established theory-driven models of the Fe|| Fe₃O₄ interface. Using X-ray PDF measurements for corroded Fe samples, and employing our newly-developed NMF analysis, we extract the experimental interface PDF (‘iPDF’) for this same system. We find excellent agreement between theory and experiment.

[1] Baraff, G. A., Appelbaum J. A. & Hamann, D. R. (1977) Phys. Rev. Lett. 38, 237.

[2] Harrison, W. A., Kraut, E. A., Waldrop J. R. & Grant R. W. (1978) Phys. Rev. B 18, 4402.

[3] Goodwin, A. L. (2019) Nat. Commun. 10, 4461.

[4] Lee, D. D. & Seung, H. S. (1999) Nature 401, 788.

[5] Geddes, H. S., Blade, H., McCabe, J. F., Hughes, L. P. & Goodwin, A. L. (2019) Chem. Commun. 55, 13317.

Complementary to x-ray diffraction patterns that represent the crystal lattice in Q space, the atomic pair distribution function (PDF) describes the structure of a material as a histogram of interatomic distances r in real space. The total scattering (TS) approach that enables PDF analysis requires that scattering data is collected over a wide Q range of the order of 20 Å^-1 and subsequent Fourier transformation of the entire scattering pattern into direct space. While TS at high-energy beamlines has become a standard routine for bulk-type samples, the unfavorable thickness ratio of a thin film (nanometer regime) to its substrate (micrometer regime) limits the detectability of the film signal in simple transmission geometry as described e.g. in Ref. [1]. Therefore, we applied the high-energy surface diffraction technique established for single-crystal surfaces [2] to less ordered films and thus pushed the capabilities for PDF analysis of thin films to unprecedented limits in terms of minimum thickness and time resolution. [3,4] Besides polycrystalline and textured metal and oxide layers, we studied amorphous and naocrystalline thin films. By careful data treatment, we successfully derived PDFs of comparable data quality from different HfO₂ films with thicknesses down to 15 nm independent on their degree of ordering with domain sizes between ~5 and >30 Å. All films were deposited on fused silica which provides an easily scalable background to subtract from the sample data to isolate the film signal. Real thin film devices e.g. for electronic applications, however, typically consist of multiple layers, and the film growth is largely affected by the nature of the underlying layer. Therefore, we further developed grazing incidence total scattering towards a depth-resolving method by scanning the incidence angle. In this way, the technique provided insight into the structure of different types of bilayer samples studied for their use e.g. in next-generation computer memory applications. PDFs were successfully extracted from the individual layers of different combinations and stackings of amorphous and crystalline materials exhibiting high and low (electron) density and, hence, x-ray scattering power from TiO₂ to Pt [5]. As thermal treatment is an essential part of thin film device manufacturing, we are developing a laser-interferometer based system that, beyond data collection during isothermal heat-treatment as applied in [4], enables following structural changes during variable-temperature processes up to several hundred degrees. Fig. 1 shows data from the proof-of-concept experiment on a 30 nm HfO₂ thin film deposited by chemical vapor deposition in an amorphous state, crystallized in situ while continuously acquiring TS data.

The antiferromagnetic semiconductor MnTe has recently attracted significant attention as both a high-performance thermoelectric and a candidate material for spintronics. The magnetic properties of MnTe play a crucial role in both of these technological applications. MnTe has a hexagonal layered structure in which magnetic Mn2+ spins order ferromagnetically within the plane and antiferromagnetically between the planes below TN = 307 K. Above TN, robust short-range magnetic correlations survive to high temperature. It has been shown that these short-range correlations are a significant contributor to the high thermoelectric figure of merit zT in MnTe through a mechanism known as paramagnon drag. Here, we present comprehensive atomic and magnetic pair distribution function (PDF) analysis of neutron total scattering data collected from pure and doped MnTe powders, together with three-dimensional magnetic PDF data obtained from a single crystal of MnTe. These complementary data sets allow us to track in detail the evolution of the magnetic correlations from the long-range ordered state at low temperature to the short-range ordered state at high temperature. We present real-space magnetic models that reproduce the observed mPDF patterns with quantitative accuracy and discuss the significance of these results in the context of existing work on MnTe.

The local structure of NaTiSi₂O₆ is examined across its Ti-dimerization orbital-assisted Peierls transition at 210 K. An atomic pair distribution function approach evidences local symmetry breaking pre-existing far above the transition. The analysis shows the dimers evolve on heating into a short-range orbital degeneracy lifted (ODL)[1] state of dual orbital character, present up to at least 490 K. The ODL state is correlated over the length scale spanning ~6 sites of the Ti zigzag chains. Our results imply that the ODL phenomenology extends to strongly correlated electron systems.

The material a-RuCl₃ has been the subject of intense study for the past few years owing to the expectation that it exhibits competing uniaxial exchange interactions characteristic of what are now termed Kitaev materials [1]. coordinated Ru³⁺ ions form a honeycomb lattice with layers weakly bonded via van der Waals interactions. The ease of formation of stacking faults and domains has made a definitive determination of the low temperature crystallographic space group difficult since many possible arrangements of the layers are energetically similar. In the absence of a magnetic field single crystals with few stacking faults show a phase transition near a Neel temperature T_N = 7 K to an antiferromagnetic structure that has zigzag order in a single plane and a 3-fold out of plane periodicity [2,3]. The introduction of stacking faults results in a structure with a 2-layer periodicity with T_N = 14 K [2,3]. The phase diagram in the presence of external in-plane magnetic field perpendicular to a Ru-Ru bond has not been fully resolved, but some features are clear, including a transition to an ordered state with a different layered periodicity near 6 Tesla and the complete suppression of zigzag order above roughly 7.5 Tesla [4,5,6]. Substitution of non-magnetic Ir4+ for Ru3+ also suppresses the zigzag order [7].This talk discusses these results in the context of measurements of the magnetic excitations, and the possible presence of a quantized thermal Hall effect and other interesting phenomena.

[1] Takagi, H., Takayama, T., Jackeli, G., Khaliullin G., & Nagler,S.E. (2019). Nature Reviews Physics 1, 264. [2] Banerjee, A. et al. (2016), Nature Materials 15, 733.

[3] Cao, H.B. A. et al. (2016), Physical Review B 93, 134423.

[4] Banerjee, A. et al. (2018), NPJ Quantum Materials 3, 8.

[5] Balz, C. et al. (2019), Physical Review B 100, 060405(R).

[6] Balz, C. et al. (2021), Physical Review B 103, 174417.

[7] Lampen-Kelly P. et al. (2017), Physical Review Letters 119, 237203.

The research discussed here used resources at the High Flux Isotope Reactor and Spallation Neutron Source, DOE Office of Science User Facilities operated by the Oak Ridge National Laboratory.

A central line of inquiry in condensed matter science has been to understand how the competition between different states of matter give rise to emergent physical properties. Perhaps some of the most studied systems in this respect are the hole-doped LaMnO3 perovskites, with interest in the past three decades being stimulated on account of their colossal magnetoresistance (CMR). However, phase segregation between ferromagnetic (FM) metallic and antiferromagnetic (AFM) insulating states, which itself is believed to be responsible for the colossal change in resistance under applied magnetic field, has until now prevented a full atomistic level understanding of the orbital ordered (OO) state at the optimally doped level. Here, through the detailed crystallographic analysis of the hole-doped phase diagram of a prototype system, we show that the superposition of two distinct lattice modes gives rise to a striped structure of OO Jahn-Teller active Mn3+ and charge disordered (CD) Mn3.5+ layers in a 1:3 ratio. This superposition leads to a cancellation of the Jahn-Teller-like oxygen atom displacements in the CD layers only at the 3/8th doping level, coincident with the maximum CMR response of the manganties. Furthermore, the periodic striping of layers containing Mn3.5+, separated by layers of fully ordered Mn3+, provides a natural mechanism though which long range OO can melt, a prerequisite for the emergence of the FM conducting state. The competition between insulating and conducting states is seen to be a key feature in understanding the properties in highly correlated electron systems, many of which, such as the CMR and high temperature superconductivity, only emerge at or near specific doping values.

Orbital molecules are clusters of transition metal cations formed by orbital- and charge-ordering in systems with direct d-d interactions [1]. Vanadium oxides exhibit a particularly rich variety of orbital molecule states, notably the V-V dimerisation that accompanies the metal-insulator transition in VO₂ [2]. In vanadium oxide spinels such as AlV₂O₄ and GaV₂O₄ the 3D connectivity of V-V nearest neighbours allows larger orbital molecules to form, and these also persist into a hidden high-temperature disordered state [3].

Not all AV₂O₄ spinels have orbital molecule ground states, but as the formation of V-V bonds is associated with marked lattice distortions we have employed synchrotron X-ray powder diffraction and pair-distribution function analysis to determine the structural and electronic requirements for V-V bonding to be stabilised. Studying the Zn_xGa_1−xV₂O₄ family of materials revealed that, whilst the long-range order of orbital molecules in the ground state of GaV₂O₄ is highly sensitive to A-site substitution, local V-V bonding interactions are stable to x > 0.75 before the ground state of ZnV₂O₄, which is orbitally ordered but without V-V bonding, emerges [4]. Furthermore, we have found a monoclinic distortion coincident with the reported pressure-driven metal-insulator transition in LiV₂O₄ that suggests it to be the result of orbital-molecule formation [5]. Overall, we have determined that the formation and ordering of orbital molecules bonds in AV₂O₄ spinels is principally dependent on the V-V nearest-neighbour distance.

[1] Attfield, J. P. (2015). APL Mater. 3, 041510.

[2] Goodenough, J. B. (1971). J. Solid State Chem. 3, 490.

[3] Browne, A. J., Kimber, S. A. J. & Attfield, J. P. (2017). Phys. Rev. Mater. 1, 052003(R).

[4] Browne, A. J. & Attfield, J. P. (2020). Phys. Rev. B 101, 024112.

[5] Browne, A. J., Pace, E. J., Garbarino, G. & Attfield, J. P. (2020). Phys. Rev. Mater. 4, 015002.

Perovskites are one of the most prevalent classes of functional materials and are already known to exhibit a wide range of properties, including ferroelectricity, superconductivity and magnetism amongst others [1]. Many traditional perovskites have poor toxicity and sustainability; however, the inclusion of organic components could help alleviate these issues and provide greater structural diversity. Several examples of hybrid perovskites with interesting properties have already been reported with the inclusion of complex anions such as cyanides, formates and azides on the X site of the perovskite [2]. Whilst the oxalate ligand has already been extensively used in coordination polymers, its use in perovskite materials has only recently been reported in the compound KLi₃Fe(C₂O₄)₃ [3], the compound exhibits simultaneous 1:3 ordering on both the A and B sites of the perovskite (Figure 1). In order to gain a more detailed understanding of this structure type, a series of compounds with the general formula A^ILi₃M^II(C₂O₄)₃ where A = K⁺, Rb⁺, Cs⁺ and M = Fe²⁺, Co²⁺, Ni²⁺ have been synthesised and characterised [4].

Figure 1. Comparison of hypothetical cubic perovskite with 1:3 cation ordering at the A and B site (left) and the corresponding crystal structure of the perovzalates (right). Li octahedra blue, M octahedra brown and A cation purple.

1 J. P. Attfield, P. Lightfoot and R. E. Morris, Dalt. Trans., 2015, 44, 10541–10542.

2 G. Kieslich and A. L. Goodwin, Mater. Horizons, 2017, 4, 362–366.

3 W. Yao, Y. Guo and P. Lightfoot, Dalt. Trans., 2017, 46, 13349–13351.

4 R. Clulow, A. J. Bradford, S. L. Lee and P. Lightfoot, Dalt. Trans., 2019, 48, 14461–14466.

Keywords: Perovskite; Hybrid materials; Coordination polymers

We would like to thank the EPSRC for a doctoral studentship to R. Clulow (DTG012 EP/K503162-) and the University of St Andrews for a doctoral studentship to A. J. Bradford

The Aurivillius materials are well known for their ferroelectric properties[1] and associated structural distortions.[2] They form a class of layered perovskite-related phases with general formula Bi₂A_n-1B_nX_3n+3 (X is usually oxide, but halides are also known), with structures built up from alternating fluorite-like [Bi₂O₂]²⁺ layers and [A_n‑1B_nX_3n+1]^2- perovskite-like layers. The search for magnetoelectrics, with coupled magnetic and ferroelectric order, has motivated investigations to introduce magnetic ions into the B cation sites. However, this has been challenging and the concentrations of magnetic B cations in Aurivillius oxides is typically low.[3-5] Redirecting research away from oxides and towards mixed-anion systems, including Aurivillius oxyfluorides, opens up a wider compositional range, as well as the possibility of tuning structure and properties by anion order.[6, 7]

This presentation describes work on n = 1 Aurivillius oxyfluorides including Bi₂TiO₄F₂ and Bi₂CoO₂F₄. Our symmetry analysis[8] of possible anion-ordered structures highlights the challenges of packing polar heteroanionic units to break inversion symmetry, as well as means by which this might be achieved for Bi₂TiO₄F₂. We also explore methods to determine anion ordering in materials with anions with similar scattering lengths.[9]

Increasing the fluoride content in these oxyfluorides gives access to phases with lower oxidation states for B cations, and the report of Bi₂CoO₂F₄, with long-range magnetic order of the Co²⁺ sublattice,[10] motivated our investigation using neutron powder diffraction. We’ve explored its nuclear structure and in particular, the anion sublattice and structural distortions, and determined its magnetic structure.[11] This gives insight into its physical properties and opens the door to designing and preparing new multiferroics.

[1] de Araujo, C. A. P.; Cuchiaro, J. D.; McMillan, L. D.; Scott, M. C.; Scott, J. F., (1995), Nature 374, 627-629.

[2] Guo, Y. Y.; Gibbs, A. S.; Perez-Mato, J. M.; Lightfoot, P., (2019), Iucrj 6, 438-446.

[3] Keeney, L.; Downing, C.; Schmidt, M.; Pemble, M. E.; Nicolosi, V.; Whatmore, R. W., (2017), Scientific Reports 7.

[4] Giddings, A. T.; Stennett, M. C.; Reid, D. P.; McCabe, E. E.; Greaves, C.; Hyatt, N. C., (2011), Journal of Solid State Chemistry 184, 252-263.

[5] McCabe, E. E.; Greaves, C., (2005), Journal of Materials Chemistry 15, 177-182.

[6] Charles, N.; Saballos, R. J.; Rondinelli, J. M., (2018), Chemistry of Materials 30, 3528-3537.

[7] Kageyama, H.; Hayashi, K.; Maeda, K.; Attfield, J. P.; Hiroi, Z.; Rondinelli, J. M.; Poeppelmeier, K. R., (2018), Nature Communications 9.

[8] Campbell, B. J.; Stokes, H. T.; Tanner, D. E.; Hatch, D. M., (2006), J. Appl. Cryst. 39, 607-614.

[9] Giddings, A. T.; Scott, E. A. S.; Stennett, M. C.; Apperley, D. C.; Greaves, C.; Hyatt, N. C.; McCabe, E. E., (2021), in preparation.

[10] Vagourdi, E. M.; Mullner, S.; Lemmens, P.; Kremer, R. K.; Johnsson, M., (2018), Inorganic Chemistry 57, 9115-9121.

[11] Scott, E. A. S.; Vagourdi, E. M.; Johnsson, M.; John, F.; Cascos, V. A.; Pickup, D. M.; Chadwick, A. V.; Zhang, W.; Halasyamani, P. S.; McCabe, E. E., (2021), in preparation.

Heusler alloys close to stoichiometric Ni₂MnGa undergo a sequence of diffusionless, displacive phase transformations from parent cubic austenite to various martensitic or ferroelastic phases, modulated 10M and 14M (also marked 5M and 10M) and non-modulated (NM) tetragonal phase depending on composition. Often the cascade of intermartensitic transformations (10M-14M-NM) is observed with decreasing temperature or with increasing mechanical stress [1]. Owing to the ferromagnetic state and highly mobile twin boundaries in modulated phases, the relatively weak magnetic field can induce the reorientation of ferroelastic domains via twin boundary motion [2]. This results in giant magnetic-field-induced strain up to 12% in single crystal [3] called magnetic shape memory (MSM) effect. Although the martensitic transformation is relatively well understood, the nature of intermartensitic transformation (IMT) is still disputed. One reason is that even the character of modulated phases is not settled [4-6]. Understanding the IMT can provide some clue to the character of modulated phases and has also practical impact as the IMT limits the operational range of the MSM effect. Although many diffraction studies were performed on polycrystalline samples only little neutron research has been done on single-crystals. Regarding the complex nature of the modulated phases and continuing discussion about their character (nanotwinning v. harmonic modulation) [4-6], only single crystalline studies represent the proper way in attempt to understand the 10M-14M intermartensitic transformation. The neutron diffraction as bulk method is particularly suitable for direct comparison with magnetic [7] and transport measurements [6].

Here we present study of 10M-14M transformation by neutron diffraction using the D9 and D10 single-crystal four-circle diffractometers and CYCLOPS (neutron Laue single-crystal diffractometer) in ILL Grenoble. The Laue method allowed continuous tracing of the transition and broader survey of the reciprocal space with temperature, revealing any changes in crystal orientation and newly occurring twinning in transformed phase. Additional laboratory X-ray diffraction using rotating anode diffractometer provide further insight and better precision. The q-scans measured at different temperatures across the transition revealed the details of the modulation, pointing to its nanotwinning character. Fine features in the q-scans suggested the traces of 10M within 14M phase in the temperature well below the IMT. The structural changes indicated by the diffraction were related to the changes of magnetic properties. In presentation, we will also look on preference of the 14M phase in epitaxial thin films compared to bulk single crystals.

[1] Ullakko, K., Huang, J. K., Kantner, C. & Handley, R. C. O. (1996) Appl. Phys. Lett. 69, 1966–8.

[2] Kellis, D., Smith, A., Ullakko, K. & Müllner, P. (2012) J. Cryst. Growth 359, 64-68.

[3] Heczko, O., Kopecký, V., Sozinov, A. & Straka, L. (2014) Appl. Phys. Lett. 103, 198-211.

[4] Straka, L., et al. (2011) Acta Mater. 59, 7450–63.

[5] Seiner, H., Straka, L. & Heczko, O. (2013) J. Mech. Phys. Solids 64, 072405.

[6] Veřtát, P., et al. (2021) J. Phys.: Condens. Matter, accepted, https://doi.org/10.1088/1361-648X/abfb8f

[7] Ge, Y. et al., "Transitions between austenite and martensite structures in Ni₅₀Mn₂₅Ga₂₀Fe₅ thin foil", available at: http://dx.doi.org/10.2139/ssrn.3813433

This work was supported by Operational Programme Research, Development and Education financed by the European Structural and Investment Funds and the Czech Ministry of Education, Youth and Sports, project number SOLID21 CZ.02.1.01/0.0/0.0/16_019/0000760. P.V. thanks for the support by the Grant Agency of the Czech Technical University in Prague, grant number SGS19/190/OHK4/3T/14. We acknowledge the Institut Laue-Langevin and the project LTT20014 financed by the Ministry of Education, Youth and Sports, Czech Republic, for the provision of neutron radiation facilities.

We have investigated the magnetic and electronic structures of crystalline dimethylammonium copper formate [(CH₃)₂NH₂]Cu(HCOO)₃; a model compound that belongs to a wide class of hybrid organic-inorganic perovskites. We present the results of a combined experimental approach, where neutron diffraction and magnetisation measurements were used to solve the ground state magnetic structure in which the same ligand mediate antiferromagnetic and ferromagnetic interactions, while electron charge density distribution and orbital occupancy were determined by high-resolution x-ray diffraction [1]. The latter provided a microscopic analysis of the chemical bonding from which we established a detailed correlation between the structural, electronic, and magnetic properties of [(CH₃)₂NH₂]Cu(HCOO)₃, demonstrating the primary role of Cu-O bonding in establishing the nature of the exchange. Our results elucidate the mechanism of magnetic exchange mediated by formate anions, from which we examine the applicability of foundational theories of purely inorganic perovskites and define characteristics that the ligands should meet to support the use of the Goodenough-Kanamori-Anderson (GKA) rules [2]. The derived criteria for the applicability of GKA rules where used to predict the magnetic structure, then verified experimentally, of hybrid perovskites including different ligands, such as [(CH₃)₂NH₂]Cu(HCOO)₂(NO₃) and Cu(HCOO)₂(pyrimidine). Charge density analysis enabled us to account for qualitative and quantitative differences in the superexchange mediated by formate, nitrate and pyrimidine.

[1] R. Scatena, R. D. Johnson, P. Manuel, P. Macchi, J. Mater. Chem. C 2020, 8, 12840–12847.

[2] S. V. Streltsov, D. I. Khomskii, Uspekhi Fiz. Nauk 2017, 187, 1205–1235.

This talk will underscore the importance of developing fast Quantum Crystallographic (QCr) [1] approaches to accurately calculate the electric fields and their associated electrostatic potentials of large molecules such as proteins.

Crystallographic structures of ATP synthase from five species have been used to calculate (approximately) and compare their intrinsic electrostatic potentials and fields [2-4]. Striking consistent patterns (and differences) in the topographies of these scalar and vector fields are uncovered across the five studied ATP synthases [2]. The role of these fields in the biological function of ATP synthase will be discussed within the context of Mitchell’s chemisomotic theory.

Our calculations suggest that, due to the intrinsic field of ATP synthase itself, the standard equation of chemiosmotic must be augmented by including a term that accounts for the contribution to ΔG of the difference in ATP synthase’s electrostatic potential, ΔΨ_ATPase, between the points of entry and exit of the protons in the mitochondrion. With this inclusion, our proposed amended equation for the chemisomotic ΔG, in standard notation, becomes [2-4]:

ΔG = ΔG_chem.+ ΔG_elec.+ (ΔG_ATPase (NEW TERM)) = 2.3 nRT ΔpH + nFΔΨ + nFΔΨ_ATPase

Our results can be summed-up into assigning two separate but complementary roles to ATP synthase ^[2]:

(1) Its putative role, and that is the catalysis (i.e. lowering the ΔG^‡) of the reaction: ADP + P_i ↔ ATP + H₂O.

(2) A novel role, that is, of altering the ΔG of the reaction of translocation of protons from the inter-membrane gap in the mitochondrion to the mitochondrial matrix, i.e. the reactions: H⁺_{inter-membrane space} ↔ H⁺_{mitochondrial matrix}.

Said differently, due to the enzyme’s very structure and due to the chemiosmotic origin of the free energy it harnesses, ATP synthase functions over an above its role as an enzyme and is more than strictly a biological catalyst.

The crucial role played by the enzyme’s own electric properties calls for their accurate and fast determinations especially with the advent of QCr [1]. An example of such approaches is the Kernel Energy Method [5-6] fragmentation whereby, given a molecular geometry, one can perform quantum calculations on fragments and obtain an approximate total electrostatic potential of the full molecules.

Since this is a part of a larger project, time permitting, the talk may touch upon some of the hot current open questions such as the one we term “Mitochondrion Paradox” [7-10].

[1] Genoni, A. ; Bucinskż, L.; Claiser, N.; Contreras-Garcia, J.; Dittrich, B.; Dominiak, P. M.; Espinosa, E.; Gatti, C.; Giannozzi, P.; Gillet, J.-M.; Jayatilaka, D.; Macchi, P.; Madsen, A. Ų.; Massa, L.; Matta, C. F.; Merz Jr., K. M.; Nakashima, P.; Ott, H.; Ryde, U.; Scherer, W.; Schwarz, K.; Sierka, M.; Grabowsky, S. (2018) Chem. Eur. J. 24, 10881-10905.

[2] Vigneau, J. N.; Fahimi, P.; Ebert, M.; Cheng, Y.; Tannahill, C.; Muir, P.; Nguyen-Dang, T.-T.; Matta, C. F. (2021) Submitted, in review.

[3] Cheng, Y. (2019). A Computational Investigation of the Intrinsic Electric Field of ATP Synthase (M.Sc. Thesis); Saint Mary's University: Halifax, Canada.

[4] Matta, C. F. (2016). Dipolar field of ATP Synthase: Unpublished results presented in a number of seminars.

[5] Huang L.; Massa, L.; Karle, J. (2010). Chapter 1 in: Quantum Biochemistry: Electronic Structure and Biological Activity, Vol. I; Matta, C. F. (Ed.), Wiley-VCH: Weinheim, 2010.

[6] Polkosnik, W.; Matta, C. F.; Huang, L.; Massa, L. (2019). Int. J. Quantum Chem. 119, e26095.

[7] Fahimi, P. ; Matta, C. F. (2021). Phys. Biol. 18 , in press (DOI: 10.1088/1478-3975/abf7d9).

[8] Fahimi, P. ; Matta, C. F. (2021) Submitted, in review.

[9] Nasr, M. A.; Dovbeshko, G. I.; Bearne, S. L.; El-Badri, N.; Matta, C. F. (2019). BioEssays 41, 1900055.

[10] Matta, C. F.; Massa, L. (2017). J. Phys. Chem. A 121, 9131-9135.

Melt-quenched glasses from metal-organic frameworks (MOF) represents a new class of hybrid functional materials, which have generated a lot of attention amongst the material science community due to their novel short-range structures and potential applications such as gas-mixture separations, ion conductivity, etc [1]. To improve the thermal stability window of the liquid phase of MOFs, substantial efforts are directed to lower the melting temperature of the crystalline MOF state [2, 3]. However, in this context, the relationship between chemical bonding and melting/decomposition of MOFs is still unexplored.

In this work, we compare the electron density distribution of two isostructural Zeolitic Imidazole Framework (ZIF) molecules-meltable Zn-ZIF-zni with Co-ZIF-zni that undergoes thermolysis, using high-resolution synchrotron single-crystal X-ray diffraction data measured at 25 K. Several ZIFs such as ZIF-4, ZIF-1, ZIF-3, ZIF-zeg, ZIF-nog undergo thermal amorphization and recrystallization to ZIF-zni prior to melting/decomposition [4]. Charge density analysis along with derived topological parameters based on Bader’s QTAIM theory [5] shows that Zn‒N bonds are primarily closed shell ionic in nature and weaker in strength. On the other hand, Co‒N bonds are dominated by polar covalent interactions with significant electron density accumulation in bonding region and distinct π-backbonding features (Fig. 1).

In situ temperature dependent Raman spectroscopy (300 K-773 K) revealed a greater degree of bond weakening in the imidazolate ligands of Co-ZIF-zni during heating. In addition, variable temperature crystallography (25 K-400 K) confirmed that Zn-ZIF-zni are less prone to framework distortion in comparison to a more rigid framework in Co-ZIF-zni. To further validate the role of metal‒ligand bonds on thermal behavior of these ZIF compounds, for the first time we prepared a set of eight novel solid solutions-Co_xZn_1-x-ZIF-zni where mole fraction (x) of Co ranges from 0.4 to as low as 0.003. Using differential scanning calorimetry (DSC)/ thermogravimetric analysis (TGA), we observed that a presence of very low quantity (~4%) of doped Co in Zn-ZIF-zni lattice results in thermal decomposition of the crystal framework. We identified this phenomenon as ‘butterfly effect’ of Co‒N bonds on thermal stability of these solid solution MOFs.

[1] Bennett, T. D. & Horike, S. (2018). Nat. Rev Mat. 3, 431-440. [2] Frentzel-Beyme, L., Kloß, M., Kolodzeiski, P., Pallach, R. & Henke, S. (2019). J. Am. Chem. Soc. 141, 12362-12371. [3] Hou, J., Ríos Gómez, M. L., Krajnc, A., McCaul, A., Li, S., Bumstead, A. M., Sapnik, A. F., Deng, Z., Lin, R., Chater, P. A., Keeble, D. S., Keen, D. A., Appadoo, D., Chan, B., Chen, V., Mali, G. & Bennett, T. D. (2020). J. Am. Chem. Soc. 142, 3880-3890. [4] Bennett, T. D., Keen, D. A., Tan, J.-C., Barney, E. R., Goodwin, A. L. & Cheetham, A. K. (2011). Angew. Chem. Int. Ed. 50, 3067-3071. [5] Bader, R. F. (1990). Atoms in molecules. Wiley Online Library.

Thermoelectric materials are able to interconvert thermal and electrical energy, and thus offer the potential to harvest waste heat through solid-state devices. A particularly interesting class of thermoelectric materials are the cubic intermetallic Half-Heusler semiconductors with XYZ stoichiometry, which show promising high temperature thermoelectric properties commonly attributed to the high degeneracy of carrier pockets in the band structure and weak electron-phonon coupling.

Half-Heuslers crystallize with YZ and XY forming tetrahedrally coordinated zinc blende networks, and XZ forming a rock salt network. Stable stoichiometric Half-Heuslers have valence electron counts of 8 or 18, which has led to interpretation of their bonding and properties within Zintl chemistry [1]. Applying Zintl chemistry to Half-Heuslers, the electroposive cation, Xⁿ⁺, donates all its valence electrons to the covalently bonded [YZ]^n- polyanion, which then fulfils the 8- or 18-electron rule. Thus, ionic and covalent bonding patterns coexist and there is a clear distinction between the bonding within the polyanion and the bonding between formal cation and polyanion. Zintl chemistry is often applied for engineering thermoelectric materials, and for Half-Heuslers, it gives rise to predictions regarding the electronic structure and defect chemistry.

Expanding on previous investigations on chemical bonding in Half-Heuslers [2,3], we present the results of computational real space chemical bonding analysis using Bader’s quantum theory of atoms in molecules and delocalization indices on a range of Half-Heusler semiconductors. This shows strong deviations from predictions from Zintl chemistry for transition metal based materials and reveal interesting relations between chemical bonding and thermoelectric and response properties [4].

We construct a map of chemical bonding in Half-Heuslers based on real space indicators [5], onto which we map important calculated thermoelectric properties. This reveals that strong covalent bonding between formal cation and polyanion results in increased carrier pocket degeneracies, and therefore improved thermoelectric properties. Thus, the materials least in line with the commonly applied Zintl concept are in fact the ones with the best thermoelectric properties.

This works extends our previous studies showing the inability of Zintl chemistry to explain the unexpected isotropic properties in thermoelectric Mg₃Sb₂ [6], and thus presents a critical view on the simplistic chemical concepts too often applied for rational materials design. Furthermore, it highlights the potential of applying tools from chemical bonding analysis and quantum crystallography for materials design.

[1] Zeier, W. G., Schmitt, J., Hautier, G., Aydemir, U., Gibbs, Z. M., Felser, C., Snyder, G. J. (2016). Nat. Rev. Mater. 1, 16032

[2] Bende, D., Grin, Y., Wagner, F. R. (2014). Chem. Eur. J. 20, 9702-9708

[3] Bende, D., Wagner, F. R., Grin., Y. (2015). Inorg. Chem. 54, 3970-3978

[4] Tolborg, K., Iversen, B.B. (2021). Submitted

[5] Raty, J. Y., Schumacher, M., Golub, P., Deringer, V. L., Gatti, C., Wuttig, M. (2019). Adv. Mater. 31, 1806280

[6] Zhang, J., Song, L., Sist, M., Tolborg, K., Iversen, B. B. (2018). Nat. Commun. 9, 4716

Quantitative convergent-beam electron diffraction (QCBED) has become established as a highly accurate and precise means of measuring bonding electrostatic potentials and electron densities [1]. To date, it has almost exclusively been performed using the Bloch-wave electron scattering formalism [2], which requires 3-dimensional periodicity throughout the scattering volume. This has restricted QCBED to bonding measurements in homogeneous, single-phased crystalline materials (just like X-ray diffraction).

The multislice formalism [3] for electron scattering dispenses with the requirement of periodicity in the direction of the electron beam. Furthermore, electron probe sizes are typically of order 1 nm in dimension for QCBED, rendering even very highly curved features in nanostructured materials locally planar relative to the electron probe. This means that nanostructures that share a crystallographically coherent interface with the surrounding matrix in which they are embedded, could in principle be analysed by multislice-based QCBED. Add to this the routine sub-nanometre precision in positioning electron probes in transmission electron microscopes, and there is the potential to map bonding charge density as a function of position in nanostructured materials for the first time.

We are attempting to measure bonding electron densities within a number of different nanostructures and also across their interfaces with the surrounding matrix material, using QCBED based on the multislice formalism. We will present some early results from several nanostructured materials such as those shown in Fig. 1 below.

Figure 1. High angle annular dark field scanning transmission electron microscopy of an Al-Cu alloy [4] (a); a (ZnO)_kIn₂O₃ (k = 5) thermoelectric oxide superlattice [5] (b); an Al-Cu-Sn alloy containing Sn-coated voids [6] (c & d). The figure also presents a CBED pattern (e) from the material in part b and a CBED pattern (f) taken through a void like the one shown in parts c and d.

[1] Nakashima, P. N. H., Smith, A. E., Etheridge, J. & Muddle, B. C. (2011). Science 331, 1583.

[2] Bethe, H. A. (1928). Ann. Phys. (Berlin) 392, 55.

[3] Cowley, J. M. & Moodie, A. F. (1957). Acta Cryst. 10, 609.

[4] Bourgeois, L., Zhang, Y., Zhang, Z., Chen, Y. & Medhekar, N. V. (2020). Nature Commun. 11, 1248.

[5] Liang, X. & Clarke, D. R. (2018). J. Appl. Phys. 124, 025101.

[6] Tan, X., Weyland, M., Chen, Y., Williams, T., Nakashima, P. N. H. & Bourgeois, L. (2021). Acta Mater. 206, 116594.

We thank the Monash Centre for Electron Microscopy where all data were collected. Many thanks to A/Prof. Matthew Weyland and Prof. Joanne Etheridge for their expertise. We thank the Australian Research Council for funding (DP210100308).

Transition metal (TM) bound hydrides can be used as hydrogen storage materials, they also play a key role in processes of catalysis, energy conversion, and search for superconductivity. However, they are very difficult to study with the method of X-ray diffraction, on the one hand, due to the fact that hydrogen atom has only one electron with density is usually strongly shifted towards its bonding partner, on the other hand, because its weak diffraction signal is strongly screened by scattering from the electron-rich heavy metal. Moreover, it is difficult to collect high quality, let alone high resolution X-ray diffraction data for such compounds. The availability of neutron diffraction data, which are the benchmark of hydrogen positions, is even more limited.

Recently, it was proven that Hirshfeld Atom Refinement (HAR), using aspherical atomic scattering factors, allows locating hydrogen atoms bonded to light elements based on standard resolution X-ray diffraction data with accuracy and precision very close to the one of neutron experiments [1]. This was a significant improvement compared to the most popular Independent Atom Model (IAM). Nevertheless, excluding this study, only 5 structures of complexes with TM-H bonds have been successfully refined with HAR [1,2] for only two of which a complementary neutron data set is available.

We present the results of HAR of around 11 X-ray structures of crystals of metaloorganic compounds containing hydrogen atoms bonded to heavy metals from period IV (Fe, Co, Cu, Ni), V (Nb, Ru, Rh, Sb), and VI (Os) for which also the corresponding neutron structure is available. Refinements were performed using Olex2 with the application of atomic aspherical structure factors computed with the DiSCaMB library [3], based on Hirshfeld partition of molecular electron density calculated for the central molecule embedded in a cluster of atomic charges and dipoles centered on the atomic nuclei of the surrounding molecules within the radius of 8 Å. Additionally, HARs without a cluster of multipoles were carried out with NoSpherA2 [2]. Wave functions were calculated using the DFT method, in each case B3LYP, PBE, and M062X functionals were used. All the calculations were performed also in the version including relativistic effects as implemented in the DKH2 Hamiltonian approach. Various basis sets were tested.

Overall, the DiSCaMB-HAR procedure elongates the TM-H bond lengths, bringing them closer to the neutron values [4]. The level of improvement is dependent on the quality of the experimental data and the refinement. The most prominent example of a successful refinement is the structure of a metalloorganic complex containing a Ru-H bond, which can be refined anisotropically to obtain the Ru-H distance in very high agreement with the one in the neutron structure (neutron: 1.598(3) Å, IAM: 1.56(2) Å, DiSCaMB-HAR(B3LYP/cc-pVTZ-DK): 1.593(11) Å, DiSCaMB-HAR(PBE/cc-pVTZ-DK): 1.599(11) Å). In four cases (Fe, Ru, and Rh complexes), refinement of ADPs of hydrogen atoms was feasible. In the case of the Fe complex, HAR performed with SHADE2-estimated H ADPs was feasible and was used to evaluate the H APDs refined with HAR. For two structures X-ray and neutron experiments were performed at the same temperature, thus direct comparison of HAR and neutron isotropic temperature factors of hydrogen atoms will be presented. The data sets were ranked according to various parameters describing data quality and refinement quality both for the neutron and the X-ray data sets, which lead to the final joint neutron and X-ray data-refinement quality ranking. The ranking reflects well how favorable the HAR-neutron TM-H bond length comparison is and correlates with the number of electrons in the TM. Examples showing the influence of factors such as the position in the ranking and the method of obtaining the molecular wave function on the quality of TM-H and other X-H bond lengths obtained with HAR will be presented.

In this presentation, I will highlight research opportunities and challenges in probing structural dynamics of molecular systems using X-ray Photon Correlation Spectroscopy (XPCS). The development of new X-ray sources, such as 4th generation storage rings and X-ray free-electron lasers (XFELs), provides promising new insights into molecular motion. Employing XPCS at these sources allows to capture a very broad range of timescales and lengthscales, spanning from femtoseconds to minutes and atomic scales to the mesoscale. Here, I will discuss the scientific questions that can be addressed with these novel tools for two prominent examples: the dynamics of supercooled water [1,2] and proteins [3]. Finally, I will provide practical tips for designing and estimating feasibility of XPCS experiments as well as on detecting and mitigating radiation damage.

[1] F. Perakis, K. Amann-Winkel, F. Lehmkühler, M. Sprung, D. Mariedahl, J. A. Sellberg, H. Pathak, A. Späh, F. Cavalca, D. Schlesinger, A. Ricci, A. Jain, B. Massani, F. Aubree, C. J. Benmore, T. Loerting, G. Grübel, L. G. M. Pettersson and A. Nilsson, Proc. Natl. Acad. Sci. U.S.A. 114, 8193-8198 (2017)
[2] F. Perakis, G. Camisasca, T. J. Lane, A. Späh, K. T.Wikfeldt, J. A. Sellberg, F. Lehmkühler, H. Pathak, K. H. Kim, K. Amann-Winkel, S. Schreck, S. Song, T. Sato, M. Sikorski, A. Eilert, T. McQueen, H. Ogasawara, D. Nordlund, W. Roseker, J. Koralek, S. Nelson, P. Hart, R. Alonso-Mori, Y. Feng, D. Zhu, A. Robert, G. Grübel, L. G. M. Pettersson, and A. Nilsson, Nature Comm. 9, 1917 (2018)
[3] F. Perakis and C. Gutt, Phys. Chem. Chem. Phys., 22, 19443-19453 (2020)

Additives provide a versatile strategy for controlling crystallization processes, enabling selection of properties including crystal sizes, morphologies, and structures. The additive species can also be incorporated within the crystal and even the crystal lattice itself, leading for example to enhanced mechanical properties. However, while many techniques are available for analysing particle shape and structure, it remains challenging to characterize the structural inhomogeneities and defects introduced into individual crystals by these additives, where these govern many important material properties. Here, we exploit coherent diffraction imaging methods to visualize the distribution of additives within as well as the effects additives have on the internal structure of individual calcite crystals. Highlighted are how factors including supersaturation, solution composition and additive-crystal interactions govern the distribution of additives in single crystals. Further, emphasized is the emergence of a range of complex strain and zonation patterns depending on the nature of the additive, diverging in part and locally from commonly suggested distribution models. This work contributes to our understanding of the factors that govern the structure-property relationships of crystalline materials, where a controlled utilization of additives will ultimately inform the design of next-generation materials.

Cateretê, the coherent X-ray scattering beamline at the new Brazilian synchrotron 5bent-achromat source, Sirius [1] is dedicated to coherent diffraction imaging (CDI) as well as X-ray photon correlation spectroscopy (XPCS) studies. Making the most of the coherence properties of the ultra-low emittance of the Sirius accelerator, will enable to perform 3D imaging of micrometer sized specimen down to few nanometers spatial resolution.

The Cateretê beamline is equipped with an undulator source, in a low-beta straight section, and two cryo-cooled focussing mirrors creating a 41 x 36 mm² (FWHM at 9 keV) coherent beam at 88 m from the source. The beamline operates in the 4 to 24 keV energy range using a horizontally deflecting 4-bounce crystal monochromator (4CM). Moving the 4CM laterally by a few mm, enables to operate the beamline in pink beam mode, maintaining the beam position unchanged. The experimental station is located 88 m from the source, followed by a 28 meters vacuum chamber hosting the Medipix (3k x 3k pixels²) in-vacuum detector.

The beamline, now under commissioning, will enable to perform imaging in reciprocal space, with a particular focus on in situ imaging as well as cryo-imaging experiments [2], [3]. To date, we measured and obtained the first three-dimensional reconstruction of a 6 microns cube zeolite crystal. XPCS studies of zeolite nucleation and growth have also been performed and will be presented.

An operando reaction cell, enabling to image catalysts under realistic catalytic conditions and a cryogenic sample environment are under development. The latter will allow 2D and tomographic data acquisition of specimens loaded in capillaries or flat substrates such as Si₃N₄membranes. The cryo-system is based on a low-flow cryo-cooled He gas preserving the sample stability and operates in a controlled humidity atmosphere preventing ice formation.

I will describe the Cateretê beamline and present the latest results obtained using plane-wave CDI as well as XPCS.

[1] L. Liu, N. Milas, A. H. C. Mukai, X. R. Resende, and F. H. De Sá, “The sirius project,” J. Synchrotron Radiat., vol. 21, no. 5, pp. 904–911, 2014.

[2] A. R. Passos et al., “Three-dimensional strain dynamics govern the hysteresis in heterogeneous catalysis,” Nat. Commun., vol. 11, no. 1, pp. 1–8, 2020.

[3] C. C. Polo et al., “Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography,” Sci. Rep., vol. 10, no. 1, pp. 1–11, 2020.

Acknowledgements: MCTI, CNPq, Fapesp (2014/25964-5).

Topological defects are at the heart of many intriguing phenomena in fields as diverse as biology and materials science. Ability to manipulate the topological order at will has transformative implications for nanotechnology, particularly for next generation spintronic devices, solar cells, photonics, reconfigurable electronics, catalysts, and energy and information storage. To achieve such control, we must deepen our understanding of topological textures. It is therefore essential to comprehend their nature in 3D.
While electron microscopy methods achieve very high spatial resolution even in 3D, for this they rely on destructive slicing/milling techniques that (1) induce excessive strain on the samples, potentially significantly altering the energy landscape; and (2) render time-dependent studies impossible. On the other hand, X-rays have high penetration depth that allows them to access whole-volume information and are (mostly) non-destructive, preserving the structures under study. Moreover, X-rays do not interfere with electric and magnetic fields (as well as with visible light photons), allowing studies to be performed under external influences.
In this talk, we will show how Bragg coherent diffractive imaging, with help of Landau phase-field modelling, can be extended to the studies of ferroelectric domains, polar vortices [1] and 1D strings [2] in individual nanoparticles under external electric fields. Our results show that topological structures in ferroic materials can modulate the structural phase transition driven by electric field. When analyzing projections of toroidal moment, we also observed controllable chirality, which can be applied in next generation electronics. Tracking of the domain morphology and the vortex core lines suggests that some ferroic materials feature topological structures of the same universality class as hypothetical cosmic strings. This suggests that our methodology can be applied to the studies as exciting and fundamental as cosmology. We will further discuss how the same methodology can be adapted to the studies of large-scale topological textures in photonic networks imaged using ptychographic X-ray computed tomography [3]. We will emphasize the similarities between imaged topological entities and discuss implications of next generation synchrotron sources for the field.

[1] D. Karpov, Z. Liu, T. dos Santos Rolo, R. Harder, P. V. Balachandran, D. Xue, T. Lookman, and E. Fohtung, “Three-dimensional imaging of vortex structure in a ferroelectric nanoparticle driven by an electric field”, Nat. Comm. 8, 280 (2017)[2] D. Karpov, Z. Liu, A. Kumar, B. Kiefer, R. Harder, T. Lookman, and E. Fohtung, “Nanoscale topological defects and improper ferroelectric domains in multiferroic barium hexaferrite nanocrystals”, Phys. Rev. B 100, 054432 (2019)[3] High-resolution three-dimensional imaging of topological textures in gyroid networks (manuscript in preparation).

On one hand, coherent diffraction imaging (CDI) in Bragg geometry has emerged as a unique 3D microscopy of nanocrystals thanks to 3rd generation synchrotron sources. Away from absorption edges and at space-group allowed reflections, it provides not only the electronic density, but also, encoded in the phase, the atomic displacement field with respect to the mean lattice, which in turn reveals crystal strain, defects and domains [1–3]. On the other hand, some crystal structures have crystallographic reflections which are forbidden by the space-group symmetry but can nevertheless be observed at a suitable X-ray absorption edge, due to the anisotropy of the tensor of scattering (ATS) [4]. They are several orders of magnitude weaker than allowed reflections, but the absence of Thomson scattering allows the observation of various electronic phenomena related to electronic orders (magnetic, charge, orbital), static and dynamic atomic displacements.
The new generation of synchrotron sources, such as the ESRF “Extremely Bright Source”, opens opportunities to perform CDI on such weak reflections. Here we report on the measurement of the (115) forbidden reflection of a GaN nanopillar at the Ga K edge. Sufficient statistics could be obtained in a total accumulation time of ~30 minutes for an entire rocking curve to retrieve the phase of the scattering function. Such measurement at high temperature would provide an image of the inhomogeneity of thermal motion in the crystal [5], which would be particularly interesting close to surfaces, inversion domain boundaries [3] and crystal defects. This proof-of-principle experiment demonstrates that forbidden reflections are a new opportunity for CDI with the new synchrotron sources.

[1] Robinson, I. & Harder, R. (2009). Nature Materials 8, 291.
[2] Clarke, J., Ihli, J., Schenk, A. S., Kim, Y.-Y., Kulak, A. N., Campbell, J. M., Nisbet, G., Meldrum, F. C. & Robinson, I. K. (2015). Nature Materials 14, 780.
[3] Labat, S., Richard, M.-I., Dupraz, M., Gailhanou, M., Beutier, G., Verdier, M., Mastropietro, F., Cornelius, T. W., Schülli, T. U., Eymery, J. & Thomas, O. (2015). ACS Nano 9, 9210.
[4] Dmitrienko, V. E. (1983). Acta Cryst. A 39, 29.
[5] Beutier, G., Collins, S. P., Nisbet, G., Ovchinnikova, E. N. & Dmitrienko, V. E. (2012). Eur. Phys. J. Special Topics 208, 53.

The authors ackowledge the ESRF for beamtime allocation under project number MI-1377.

A solution to the crystallographic “phase problem” was proposed by David Sayre immediately after the announcement of Shannon’ Information Theorem, requiring the diffraction to be sampled more than twice as finely as the Bragg peak spacing [1]. The implicit need for X-ray coherence has been happily solved with the development of the latest synchrotron sources, where Bragg Coherent Diffraction Imaging (BCDI) experiments are routinely performed. The fringed diffraction patterns can be oversampled so as to overdetermine the phase problem. Iterative algorithms that converge on the solution. Despite meeting all the oversampling requirements of Sayre and Shannon, current iterative phase retrieval approaches still have trouble achieving a unique inversion of experimental data in the presence of noise. We propose to overcome this limitation by employing Machine Learning in a Convolutional Neural Network model which combines supervised training with unsupervised refinement. Remarkably, our model can be used without any prior training to learn the missing phases of an image based on minimization of an appropriate “loss function” alone. We demonstrate significantly improved performance with experimental Bragg CDI data over traditional iterative phase retrieval algorithms [1,2].

The Symmetry Database (https://symmdb.iucr.org/) forms part of the online edition of International Tables for Crystallography and gives access to databases of crystallographic point and space groups. These online databases expand and complement the symmetry information provided in the print editions of International Tables for Crystallography Volume A, Space-Group Symmetry [1] or Volume A1, Symmetry Relations between Space Groups [2]. The information in the database can either be retrieved directly or generated ‘on-the fly’ using a range of programs. Help pages briefly explain the crystallographic data and the functionality of the programs. The data and programs that are currently available in the Symmetry Database are arranged into three sections:

(i) Space-group symmetry: The data in Volume A are extended to include the generators, general positions and Wyckoff positions of all 230 space groups, including the 530 settings for the monoclinic and orthorhombic space groups listed in Volume A. If data are not available for a particular setting directly, an arbitrary basis transformation can be specified and the data will be transformed to this new basis. The Wyckoff positions are specified by the Wyckoff letters, multiplicities, coordinate triplets and site-symmetry groups. Optionally, the symmetry operations of the site-symmetry groups of any point (within the unit cell or specified by its coordinates) can be calculated. Different types of notation are used for the symmetry operations: they are presented as coordinate triplets, in matrix form, using geometric symbols (indicating the type and order of the operations, and the location and orientation of the corresponding geometric elements, and screw or glide components if relevant) and as Seitz symbols. Information is also available for the Euclidean, chirality-preserving and affine normalizers of the space groups.

(ii) Symmetry relations between space groups: The maximal subgroup data given in Volume A1 are extended to subgroups of arbitrary index for all the space groups, and series of isomorphic subgroups are available for indices up to 50 for orthorhombic, tetragonal, trigonal and hexagonal space groups and for indices up to 27 and 125 for cubic space groups. Interactive contracted and complete graphs of chains of maximal subgroups, including basis transformations and origin shifts for each step, can also be generated. In addition, data for supergroups of arbitrary index of all the space groups are provided. In contrast to Volume A1, where only space-group types of supergroups are indicated, in the symmetry database each supergroup is listed individually and specified by the transformation matrix that relates the conventional bases of the group and the supergroup. The subgroup and supergroup data can be transformed to the basis of the group, left- and right-coset decomposition calculations can be carried out, and Wyckoff-position splittings can be obtained along with the relations between the coordinates of the positions within the group and subgroup.

(iii) 3D Crystallographic point groups: The data for the point groups, presented in an analogous way to the space-group data, include generators, and general and special Wyckoff positions. The data can be transformed to different settings, thus enhancing and extending the data tabulated in Volume A. Clear and instructive visualization of the symmetry elements of the crystallographic point groups and their stereographic projections, including interactive 3D polyhedra representations of idealized crystals, is also provided [3].

The Symmetry Database is available to all subscribers to the online version of International Tables for Crystallography. A Teaching Edition of the Symmetry Database, which can be used to obtain and explore the data for a selected set of space groups is also available online.

The Symmetry Database has been developed as part of an ongoing project between International Union of Crystallography, eFaber Soluciones Inteligentes SL. (Bilbao) and the Bilbao-Crystallographic-Server team. Most of the additional crystallographic data for the space groups, their subgroups and supergroups, and program algorithms have been provided by the Bilbao Crystallographic Server (www.cryst.ehu.es).

[1] International Tables for Crystallography (2016). Volume A, Space-Group Symmetry, 6th ed., edited by M. I. Aroyo. Chichester: Wiley.

[2] International Tables for Crystallography (2010). Volume A1, Symmetry Relations between Space Groups, 2nd ed., edited by H. Wondratschek & U. Müller. Chichester: John Wiley & Sons.

[3] Arribas, V., Casas, L., Estop, E. & Labrador, M. (2014). Comput. Geosci. 62, 53–61.

The ARP/wARP software provides automated model building in macromolecular crystallography and cryo-EM maps for structures of proteins, and their complexes with nucleic acids and small molecule ligands. The ARP/wARP remote service for macromolecular model building has been available since 2004 and was used to provide tens of thousands model building jobs remotely submitted by more than 4,000 users. A comprehensive description of the ARP/ARP web service, including a historical perspective will be provided. To allow the user a direct monitoring of the model building task, its progress and accumulated results are displayed graphically (e.g. the Wilson plot, the development of crystallographic R/Rfree-factors, the number of residues built) and in a tabular form as well as JavaScript-based cartoons of the built structures. The output files can also be downloaded when the job is completed. A user can rerun jobs with modified parameters and the results of these can be compared to each other. The analysis of the accumulated data and a number of take-home messages will be presented.

Macromolecular crystallography instruments around the world run more and more in a remote access or unattended configuration. This leads to less contact between humans and the hardware as well as less awareness of software and hardware states. Beamline failures that were in the past routinely reported by humans are now missed and lost in the noise of other issues. On another hand there is a need to have a chain of triggers from the beamline failure to the call out of a synchrotron staff that can assess and fix an issue. Finally, although most facilities have constant monitoring tools such has text messages or emails on catastrophic failures like loss of vacuum or cooling in the DCM, they tend to not monitor less important values due to the incapacity of a human being to deal with excessive amounts of information including false positives. Here we present a beamline monitoring software that intents to monitor EPICS PVs as well as other systems via HTTP restful interfaces, database connections or on disk file analysis and report in a configurable way to systems such as Slack, Email, Signal/WhatsApp or others. The use of a Slack bot allows update of configuration notifications as well as query some beamline states remotely before a support remote connection is required. Concepts as beamline mode as well as custom notifications for different staff members as well as a dependency chain of failures help reduce the number of notifications to a level which can be dealt with. The expectation is that this will be part of the on call / callout system, monitor the beamline live for upcoming possible problems as well as provide a log of the beamline states for the last day(s).

Machine learning can be justified only if input descriptors are crystal invariants independent of accidental choices. To use a household analogy, the average color of human clothes can be the easiest descriptor to extract from images but cannot be seriously considered for learning reliable information about people. Similarly, no properties of crystals can be reliably predicted from ambiguous parameters of a unit cell and a motif. Since crystal structures are determined in a rigid form, they should be considered equivalent modulo rigid motion or isometry, which preserves all interpoint distances. Then crystals can be justifiably distinguished only by isometry invariants that are independent of a unit cell and are preserved under any translations and rotations. Though Niggli’s reduced cell is unique, it is discontinuous under atomic perturbations, which are always present in real crystals. This continuity of invariants is important to quantify similarities between near identical crystals obtained by Crystal Structure Prediction as approximations to energy minima.

All machine learning approaches implicitly assume that a target property continuously depends on a given input, for example similar crystals should have close values of their lattice energy. We experimentally tested that the lattice energy is discontinuous with respect to the density, powder X-ray diffraction and packing similarity (root mean square deviation as computed by Mercury). For example, many crystals detected as similar by the above tools have very different energies. The AMD invariants are not only theoretically continuous under perturbations but also satisfy continuity for energy learning: we experimentally identified a distance threshold d and a constant c such that any distance between AMD invariants smaller than d guarantees an energy difference smaller than c times d.

Standard machine learning tools were trained on AMD invariants without chemical data for 10 min and predicted the lattice energy with a mean average error of less than 5KJ/mole on a CSP dataset of 5679 crystals containing about 250 atoms per unit cell.

Distances between AMD invariants are computed so fast that the pairs of all 229K organic molecular crystals from the Cambridge Structural Database were processed overnight on a modest desktop. The AMD invariants were recently extended to a complete isoset that uniquely and continuously represents any periodic crystal and allows an explicit reconstruction of a crystal.

For over 40 years, the Collaborative Computational Project Number 4 in Protein Crystallography (CCP4) has maintained, developed, and provided an integrated Suite [1] of world-class software that allows researchers to determine macromolecular structures by X-ray crystallography and other biophysical techniques.

Traditionally, the Suite is operated via CCP4i(2) graphical user interface, available for all major desktop platforms. More recent developments include interfaces that offer users the convenience of crystallographic computing on mobile devices and access to cloud-based resources. There are several good reasons for exploiting the distributed computing paradigm in crystallography.

First, cloud-based solutions have become particularly appealing given recent advances in automated structure solution methods. Such methods are demanding for both computing power and various databases, making them less convenient for offline setups.

Second, the cloud model of operations relieves researchers from the burden of maintaining software locally, providing 24/7 access to always ready, tested, and updated software setup.

Third, cloud computing streamlines data management and logistics. Collected data may be put in cloud-based projects directly from synchrotrons, bypassing offload to user devices. Cloud projects can be shared in real-time between a team of researchers working from various geographic locations. This aspect has been particularly helpful at the virtual CCP4 workshops during the pandemic.

CCP4 currently provides two interfaces for online work [2]. CCP4 Online, started from automatic Molecular Replacement service “BALBES” in 2008, is a web portal allowing users to run in the cloud the molecular replacement and experimental phasing pipelines in the CCP4 suite. In 2020, CCP4 released an advanced online platform, CCP4 Cloud, featuring a full desktop experience online. CCP4 Cloud includes an HTML5 interface for most crystallographic tasks and allows to develop and maintain structure solution projects completely online using common web browsers on any modern platform, including mobile devices.

We will discuss the latest developments, achieved results, and future directions. Providing a global computing infrastructure for protein crystallography is now a feasible task; are we ready to accept it in practice?

[1] M. D. Winn et al. Acta. Cryst. D67, 235-242 (2011)
[2] E. Krissinel, V. Uski, A. Lebedev, M. D. Winn, C. Ballard. Acta Cryst. D74: 143-151 (2018)

All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. There are many different technologies designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference; among others). Evaluation of the data from the binding experiments and its fitting is an essential step towards the quantification of binding affinities. Here, we present user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments. The modules from our data analysis platform (spc.embl-hamburg.de) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (K_ds) and thermal unfolding parameters such as melting temperatures (T_ms).

The Protein Data Bank (PDB) has grown from a small data resource for crystallographers to a worldwide resource a very broad community of researchers and educators. In this talk I will describe the history of the growth of the PDB and the role that the community has played in developing standards and policies. I also present examples of how other biophysics communities are collaborating with the worldwide PDB to create a network of interoperating data resources. This network will expand the capabilities of structural biology and enable the determination of increasingly complex structures.

Half of the world’s population is at risk of contracting malaria, which is caused by Plasmodium parasites. Despite extensive public health and biomedical measures, the incidence of malaria continues to rise, with over 200 million cases each year. A highly effective vaccine will likely be required to eradicate malaria; however, current vaccine candidates against Plasmodium falciparum have fallen short in great part because of a lack of understanding of immunity to the parasite at the molecular level. Our recent X-ray crystallography and cryoEM work has revealed structural details of antibody immunity against the malaria parasite. These molecular blueprints of parasite inhibition by antibodies are being leveraged as guides for the design of next-generation subunit vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. We conducted a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Neutron diffraction data is guiding hydrogen placement to improve docking calculations. Several hits have promising activity in solution and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors. The role of entropy in modulating binding affinity will also be discussed.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL^TIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88^TIR). Due to their crystal size, conventional crystallography proved to be challenging. However, through serial femtosecond crystallography (SFX) we were able to determine the structure of MyD88 crystals. Here, we present the SFX structure of the MyD88^TIR assembly, which revealed a biological relevant two-stranded higher order assembly arrangement of TIR domains analogous to that seen previously for MAL^TIR. Our study provides structural and mechanistic insights into TLR signal transduction¹.

¹Clabbers, M., Holmes, S. et.al. MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography. Nature Communications, accepted March 2021, DOI: 10.1038/s41467-021-22590-6

Post-translational modification of proteins regulates many biological processes. Acetyltransferase transfers acetyl groups to lysine residues on target proteins and is a major type of post-translational enzyme. General control nonderepressible5 (GCN5, also known as Kat2a) is one of the histone acetyltransferases that promote transcriptional activity.
Metazoans possess two GCN5 isoforms that arise from alternative splicing. The lower molecular weight isoform (isoform2) is similar in size and function to yeast GCN5, consisting of an acetyltransferase (AT) domain and a bromodomain at the N and C termini, respectively. Another higher molecular weight isoform (isoform 1) has an N-terminal extension. The amino acid sequence of this N-terminal extension is similar to the N-terminal domain of p300/CBP-associated factor (PCAF, also known as Kat2b) and is conserved among vertebrates. This N-terminal domain is called the PCAF_N domain.
Ubiquitination is also a post-translational modification that targets lysine residues. This modification regulates many cellular processes, including cell division and immune responses, among others. Ubiquitination is catalyzed by the sequential reaction of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which is responsible for the ligation of ubiquitin onto a substrate in conjunction with the E2.
A previous study reported that PCAF also harbors ubiquitination activity in addition to acetyltransferase activity. PCAF_N is identified as a domain that contains a ubiquitin E3 ligase activity. Although the longer isoform of GCN5 possesses the PCAF_N domain, it has not been revealed whether GCN5 functions as an E3 enzyme.
We demonstrated that GCN5 exhibited ubiquitination activity and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that the PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which could not be predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides valuable insight into not only the functional significance of GCN5 but also into ubiquitin biology.

Lectins, carbohydrate recognizing proteins, play an important role in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganisms and hosts [1]. In connection with the last-mentioned process, lectins from pathogenic bacteria can mediate the first step of infection and they are considered an important virulence factor.

Photorhabdus laumondii is an entomopathogenic bacterium, which is known for its complicated life cycle, including mutualism and pathogenicity towards two different invertebrate hosts [2]. This contribution is focused on the newly described PLL lectin family, which shares a seven-bladed beta-propeller fold. All five members of this family are highly similar to each other in primary, secondary, and tertiary structure. However, the oligomeric state of these lectins differs significantly. Members of the PLL family have been confirmed to bind multiple monosaccharides, including l-fucose and O-methylated saccharides. X-ray structures of PLL family discovered two sets of binding sites with different ligand specificity per monomer, “polar” sites and “hydrophobic” sites. Amino acids involved in the ligand-binding are highly conserved within the lectin molecule. Ligands are bound in both types of binding sites via hydrogen bonds and via CH-π interaction with aromatic residues. Lectin/saccharide interaction is mostly mediated via hydrogen bonds. However, hydrophobic sites are deepened with a hydrophobic pocket. The importance of non-polar interactions, such as CH-π interactions between aromatic amino acids and apolar part of carbohydrate molecules, was shown recently [3].

[1] Lis, H. and Sharon, N. (1998) Chem Rev. 98, 637-74.

[2] Clarke, DJ. (2020) Microbiology. 166, 335-348.

[3] Wimmerová, M., Kozmon, S., Nečasová, I., Mishra, S.K., Komárek, J., Koča, J. (2012) Plos One. e46023.

Alzheimer’s disease (AD) is a neurodegenerative disease accompanied by the accumulation of misfolded proteins. AD pathology is characterized by the extracellular amyloid plaques and the neurofibrillary tangles (NFTs). NFTs consist of paired helical filament (PHF) and abnormally phosphorylated tau is known to form the PHF.

Tau protein that exists in the brain neuron cells is important for the stabilization and elongation of the microtubules. Tau contains a microtubule-binding domain (MBD) consisting of three or four repeats of about 30 similar amino acids (R1-R4). The MBD is not only important for the binding of tau to microtubule but also plays a key role for the abnormal self-aggregation of tau. Even though the sequences of repeat peptides of MBD are similar to each other, the ability of self-aggregation of these peptides is quite different. Especially, the short regions of both ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹ that are the start sequence of R2 and R3 respectively have an important role for the tau self-aggregation.

Several therapeutic approaches targeting tau aggregation have been proposed, such as a tau aggregation inhibitor. Among tau aggregation inhibitors, a monoclonal antibody may be particularly effective because of its specificity to the target molecule. Thus, in searching for a tau aggregation inhibitor, we made a monoclonal antibody to tau (Tau2r3) using the ²⁷²GGKVQIINKKLD²⁸³ epitope peptide from the MBD in tau and prepared the Fab domain (Fab2r3) from Tau2r3. We analyzed the inhibitory function of Fab2r3 for tau aggregation and determined the tertiary structure of the Fab2r3 complex with VQIINK peptide.

The results of thioflavin S (ThS) fluorescence and TEM (negative-staining electron microscopy) measurement clearly showed that Fab2r3 inhibited the tau aggregation, and the inhibitory function of Fab2r3 seems to occur through specific binding to the VQIINK sequence by the isothermal titration calorimetry (ITC) analysis.

To elucidate this inhibition mechanism, we analyzed the tertiary structure of Fab2r3 and VQIINK peptide complex by X-ray crystallography. The VQIINK peptide makes many hydrogen bonds and two hydrophobic interactions with Fab2r3. Among these interactions, we supposed that the hydrophobic interaction has a key role for the antigen recognition of the Fab2r3.

The fusogenic avian Orthoreovirus (ARV) infection can cause considerable economic losses in the poultry industry, mostly infecting young chickens. The ARV has been associated with various disease conditions in poultry (enteric and respiratory diseases, myocarditis, hepatitis, stunting-malabsorption syndrome, and the most important one viral arthritis/tenosynovitis) [1]. The ARV are non-enveloped icosahedral particles of 85 nm external diameter with 10 dsRNA genomic segments (23.5 kb) encased within two concentric protein shells, forming the outer capsid and the core [2].

Reoviruses' RNA replication and morphogenesis occur exclusively within cytoplasmic inclusion bodies, also known as ‘viroplasms’ or viral factories (VF). VF are globular, dynamic, phase-dense, cytoplasmic inclusions lacking membranes or cellular organelles. VFs are formed by abundant viral non-structural (NS) proteins and structural proteins recruited into VF by interaction with NS proteins. NS proteins are expressed inside the infected cells but are not part of the mature virion. The two most abundant VF proteins are μNS and σNS. μNS is a 70 kDa protein that is forming viroplasm inside infected cells and attracts and associates with other viral proteins including 41 kDa RNA chaperone σNS [3]. σNS is causing specific RNA-RNA interaction between all 10 genomic segments specifically by destabilizing of RNAs helical regions [4]. There is missing information about a fashion of coupling of μNS with σNS protein needed for the understanding of viroplasm formation mechanism.

Only a low-resolution structure of σNS has been established while no structural information is available for μNS. This is chiefly due to the poor solubility of μNS and polydispersity of σNS which forms oligomers ranging dimers to octamers and RNA containing filaments [4]. To tackle these problems and obtain structural information we have generated various fusion constructs for expression, purification, and further structural study by X-ray crystallography and electron cryo-microscopy. Due to their multi-facet role in virus biology, the detailed knowledge of their structure is necessary for a better understanding of their functions and could provide the rationale for the development of new antiviral drugs.

[1] Jones R. C. (2000). Revue scientifique et technique (International Office of Epizootics), 19(2), 614–625. https://doi.org/10.20506/rst.19.2.1237.

[2] Zhang, X., Tang, J., Walker, S. B., O'Hara, D., Nibert, M. L., Duncan, R., & Baker, T. S. (2005). Virology, 343(1), 25–35. https://doi.org/10.1016/j.virol.2005.08.002.

[3] Benavente, J., & Martínez-Costas, J. (2007). Virus research, 123(2), 105–119. https://doi.org/10.1016/j.virusres.2006.09.005.

[4] Borodavka, A., Ault, J., Stockley, P. G., & Tuma, R. (2015). Nucleic acids research, 43(14), 7044–7057. https://doi.org/10.1093/nar/gkv639.

Surface layer proteins (Slp) assemble into highly regular 2D crystalline arrays and represent the outermost cell envelope in many bacteria and archaea. The Surface layer (S-layer) is composed mostly of a single (glycol)protein species and is in close contact with their surrounding. Therefore, these arrays fulfill various functions like bacterial adherence to other cells or substrates, protection against life-threatening conditions, and maintenance of the cell shape.

The S-layer of L. acidophilus consists of two proteins. SlpA is mainly expressed under normal physiological conditions, whereas SlpX expression is increased under osmotic stress. S-layer proteins have two functional regions in common: a region that is important for the attachment to the cell wall and a region responsible for the self-assembly of the S-layer array.

Our goal is to structurally characterize the S-layer proteins SlpA and SlpX of L. acidophilus and to further understand the mechanism of the self-assembly, how the two proteins interact with each other and how the attachment to the cell wall interact occurs. Since full length S-layers form insoluble 2D crystals we designed three functional protein fragments of both proteins and we obtained diffracting crystals of all. In a joint effort and in combination with various different approaches like Hg-SAD, ARCIMBOLDO at a resolution of 1.4Å, ab initio prediction with RoseTTAfold of an only beta-strand protein and molecular replacement we were able to obtain the crystal structures of all protein domains. The structures of the self-assembly regions of SlpA and SlpX show an interesting domain switch and together they suggest the mode of action how the self-assembly of the S-layer occurs.

Acknowledgments:

The X-ray experiments were performed at synchrotron-radiation facilities ESRF (ID23-1, ID30B, ID30A, ID23-2 and ID29, Grenoble, France), DESY (P11, PETRAIII, Hamburg, Germany), EMBL (P13, PETRAIII, Hamburg, Germany), Elettra (XRD1 and XRD2, Trieste, Italy) and SLS (PXI, Villigen, Switzerland). We are grateful to local scientists for providing assistance in using the beamlines. This work has been supported by the Austrian Science Fund (FWF, P29432)

PTG (protein targeting to glycogen) is a scaffolding protein that is involved in the activation of glycogen synthesis by bringing PP1 (type 1 protein phosphatase) to its substrates. It is proposed as a target for the treatment of Lafora disease (LD), a genetic disorder manifested by catastrophic teenage onset of progressive myoclonus epilepsy. In healthy neurons, PTG is downregulated by the laforin-malin complex resulting in very low glycogen production. Mutations in malin or laforin causes accumulation of PTG, which promotes glycogen synthesis by directing PP1 to glycogen synthase and glycogen phosphorylase. This results in the appearance of neurotoxic inclusion bodies formed by insoluble polyglucosans called Lafora bodies (LB), which are ultimately responsible for Lafora disease (LD). In LD mice models knocking out PTG resulted in a nearly complete disappearance of LB and resolution of neurodegeneration and myoclonic epilepsy, indicating that small molecules interfering with the PTG/PP1 interaction emerge as an excellent therapeutic strategy for LD. Up to date, there was no structural data of PTG and PTG/PP1 complex allowing for identification of potential druggable pockets. We were able to set up expression, purification and crystallization protocols of different constructs of PTG and PTG/PP1 complex for structural studies. Here we present preliminary crystallographic structures of PTG carbohydrate binding module 21 (CBM21) and PTG/PP1 complex, complemented with SAXS analysis of the complex, that will provide a valid information for the rational design of selective compounds targeting interaction of PTG with its partners.

Antibiotic resistance is usually shared between bacteria by using conjugation [1], where a bacterium transfers part of its genome to another bacterium by involving a complex machinery called a secretion system. Conjugative Type IV secretion systems (Conj-T4SSs) are one of the most prevalent ways to share DNA between prokaryotes. Integrative and conjugative elements (or ICEs) are one of the principal types of mobile genetic elements that enable horizontal transmission of genetic information. ICEs encode their own Conj-T4SSs to perform conjugation and have the particularity to be integrated into the chromosome of the host cell. While descriptions of Conj-T4SSs in Gram-negative bacteria are available from CryoEM experiments, no data exist for Gram-positive bacteria yet. In Streptococcus thermophilus, ICESt3 encodes 14 putative proteins involved in the conjugation process. Among them, the Gram-negative VirB8-equivalent protein, called OrfG, is expected to be essential in the plasma membrane part of T4SS since it is supposed to act as an interaction hub for other T4SS proteins. OrfG contains three domains, one transmembrane domain and two soluble ones. Here we present the X-Ray structures of the soluble domains of OrfG. We performed an in-depth analysis of all VirB8-like domains [2] by using existing and new multi-protein structural alignment visualization tools. One of these tools, called MPSA_Viewer, was developed in our lab and significantly improved the visual readability of multi-protein structural alignments, especially when sequence identities are very low. We also analyzed the quaternary structure of OrfG, which consists of a trimeric assembly of interwoven monomers. The trimeric organization seems specific to VirB8-like proteins of Gram-positive bacteria since two other occurrences were found in the structural database. Such intricated assemblies can be biologically relevant, as observed for instance in the prefusion conformation of the 2019-nCoV spike [3]. We also noticed that the variable spacing at the center of these Gram-positive trimers might be compatible with substrate translocation [4] if other conditions are met.

[1] Frost, L. S., Leplae, R., Summers, A.O. & Toussaint, A. (2005) Nat. Rev. Microbiol. 3, 722.

[2] Cappèle, J., Mohamad Ali, A., Leblond-Bourget, N., Mathiot, S., Dhalleine, T., Payot, S., Savko, M. Didierjean, C., Favier, F. & Douzi, B. (2021). Front. Mol. Biosci., 8, 642606.

[3] Wrapp D, Wang, N. Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S. & McLellan, J.S. (2020). Science, 367, 1260.

[4] Miletic, S., Fahrenkamp, D., Goessweiner-Mohr, N., Wald, J., Pantel, M., Vesper, O., Kotov, V. & Marlovits, T.C. (2021) Nat. Commun. 12, 1546.

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids, or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal, and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

The world is meeting the challenge of the energy crisis, which prompted searching for renewable energy resource. Brown algae are ideal feedstocks for producing biofuels, the promising renewable resource. The sea hare Aplysia kurodai is an excellent model for investigating the biofuel production process. It consumes brown algae as a staple food to release a large amount of glucose from laminarin-depolymerization by the ?-glucosidase in its digestive fluid (akuBGL). However, brown algae produced abundant secondary metabolite as a defense against the herbivores, such as phlorotannin. Phlorotannin inhibits akuBGL activity, which causes a problem in biofuel production from brown algae. Interestingly, Eisenia hydrolysis enhancing protein (EHEP) existed in the digestive fluid of Aplysia kurodai and, was found to protect akuBGL from phlorotannin-inhibition by binding with phlorotannin and precipitating¹. How EHEP bind to phlorotannin, why it can protect akuBGL from inhibition both are unknown.

In this study, we obtained EHEP and akuBGL from digestive fluid of A. kurodai. We determined the structures of EHEP in apo form and complex with an analogue of the phlorotannin, tannic acid by native-SAD method². The structures reveal that EHEP consisted of three chitin-binding domains linked by two long loops (Fig.1) and tannic acid bound at the center of EHEP. Furthermore, we determined the structure of akuBGL and showed it comprised of two GH1 (glycoside hydrolysis family 1) domains linked by the loop (Fig.2). Additionally, the docking analysis of tannic acid with akuBGL was performed, revealing the tannic acid occupies the active pocket of akuBGL. Collectively, we proposed the mechanism of EHEP that protects akuBGL from inhibition.

Human C-reactive protein (CRP) is an innate immune macromolecule of hepatic origin produced in response to inflammatory cytokines. CRP serum concentration is exploited as a clinical biomarker in humans as levels rise rapidly in response to inflammation, infection, or tissue damage [1]. CRP has opsonising abilities and roles in the inflammatory response and activation of complement.

Native human CRP consists of five identical non-covalently bound subunits. The five protomers of CRP are arranged symmetrically around a central pore, consisting of 206 amino acids folded into two antiparallel β-sheets with a flattened jellyroll topology [2]. Each protomer has a calcium dependent ligand binding site and an effector binding site on the opposite sides of the molecule. The ‘recognition’ face binds phosphocholine (PC) in a calcium-dependent manner in a ligand binding site located within a hydrophobic pocket. PC is a principal ligand for CRP; widely expressed on the surface of damaged cell membranes and distributed in lipopolysaccharides of bacteria and other microorganisms [1]. PC binding is mediated by a phosphate-calcium interaction. The opposite ‘effector’ face of CRP accommodates multiple binding sites, for C1q and immunoglobulin Fcγ receptors. The putative C1q binding site is located at the end of a cleft bordered by the pentraxin helix [3].

Although CRP is remarkably stable under physiological conditions, it has been shown that CRP can dissociate into individual subunits to form monomeric CRP (mCRP). Evidence is increasing that monomeric CRP may have a pro-inflammatory role. We have successfully dissociated CRP, in the presence of urea, into mCRP in-vitro and identified a monomeric CRP with the same reactivity as that seen in patient samples [4]. In addition to the presence of urea, the removal of calcium ions to destabilise the protein is required. Monomeric CRP has been produced via urea-induced dissociation, optimised at 3M urea over a ten-week period [4]. This CRP form retains its reversible PC-binding ability. Another form of monomeric CRP has been observed in vitro, produced during excessive denaturing conditions, requiring 8M Urea [5] which does not retain the ability to bind PC. Optimisation of production of these in vitro mCRP forms and crystallisation trials are currently underway.

The stringent response is a fundamental mechanism for bacterial survival and adaptation to a wide range of stress conditions, mediated by the synthesis and hydrolysis of signal molecules collectively referred to as alarmones [1, 2]. Bifunctional enzymes belonging to the RelA‑SpoT homologue (RSH) superfamily are responsible for the synthesis of alarmones guanosine 5'-triphosphate 3'-diphosphate (pppGpp), guanosine 5'-diphosphate 3'-diphosphate (ppGpp), and guanosine 5′-monophosphate 3′-diphosphate (pGpp), and contain both synthetase and hydrolase domains, however, with the hydrolase domain being inactive in many cases. Some organisms additionally contain monofunctional small alarmone synthetases (SAS) and small alarmone hydrolases (SAH), regulation of which has not yet been fully described [3].

Here, we present a 1.2 Å structure of the Leptospira levetii small alarmone hydrolase (SAH) and a 1.8 Å structure of Corynebacterium glutamicum small alarmone hydrolase, together with an analysis of similarities and differences with the known bifunctional Rel enzyme structures. We show that the SAH structures contain common features typical for hydrolases, such as a metal ion binding site and the highly conserved histidine–aspartate (HD) motif. Analysis of the structures using PISA surprisingly revealed they both form dimers, in contrast to earlier reports. Moreover, there is a distinct difference in the dimerization interface, despite a high degree of structure conservation between the monomers. Dimer formation was confirmed experimentally using size exclusion chromatography multi-angle light scattering (SEC-MALS) and small angle X-ray scattering (SAXS). The structures of the two small alarmone hydrolase representatives allowed for a structural analysis and comparison with known bifunctional hydrolase structures (RelSeq from Streptococcus equisimilis and RelTh from Thermus thermophilus) [4, 5]. We demonstrate that while the key residues involved in substrate coordination are clearly conserved, there are some notable differences in several secondary structure elements. Intriguingly, we find differences in the position of helices involved in the regulation of activity in bifunctional hydrolases, that adopt an unanticipated position in the SAH structures. Taken together, our data shed new light on how small alarmone hydrolases are regulated as well as how they differ from the larger bifunctional enzymes. In the future, this will help us further understand the role of monofunctional SAH enzymes and in turn bring forward a better understanding of the stringent response in bacteria.

Bacterial chromosomes contain large numbers of toxin-antitoxin (TA) systems, consisting of a gene encoding a toxic protein and a gene encoding an antitoxin which can be an RNA or a protein [1, 2]. In the largest known group of type II TA systems, vapBC, the toxin VapC is an endoribonuclease belonging to the PIN (PilT N-terminal) domain family, which is very conserved structurally and found in all domains of life. VapC is a known RNase and targets for VapCs include various tRNAs and rRNAs. The VapB antitoxin inhibits the toxin with its C terminus domain and contains a DNA binding domain in the N terminus [1].

TA systems have been implicated in bacterial tolerance to antibiotics, which can eventually lead to antibiotic resistance and it is considered a major health challenge by the WHO [3]. Previously, it was experimentally observed when a bacterial strain developed antibiotic tolerance in the presence of ampicillin during intermittent exposure, the development of tolerance increase the risk of the strain also developing antibiotic resistance [4]. In strains of Escherichia coli KLY where tolerance was observed, it was shown that mutations in a gene encoding VapB were present [4].

However, it is not known how or if TA systems are involved in creating tolerance and potentially affect antibiotic resistance. Here, we determine the structure of VapBC from E. coli KLY to 2.8 Å using x-ray crystallography. The structure is overall very similar to the previously determined structure of a VapBC complex from Shigella flexneri 2a, where the VapBC is encoded on the pMYSH6000 plasmid. This VapBC was shown to form a hetero-octameric complex containing four VapB and four VapC proteins [5]. The VapBC from E. coli KLY differs from the VapBC in S. flexneri 2a in six positions. The two VapBs have two amino acids difference and the VapCs have four amino acid differences among them.

In the near future, structures representing several VapBC complexes with naturally occurring tolerance mutations will be determined. We envisage that the information gained will allow us to hypothesize the possible functional implications of the mutations and the possible effect this could have on the bacterium and whether this could explain an antibiotic tolerant phenotype.

1. Bendtsen, K.L. and D.E. Brodersen, Higher-Order Structure in Bacterial VapBC Toxin-Antitoxin Complexes. Subcell Biochem, 2017. 83: p. 381-412.

2. Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.

3. Antimicrobial resistance. Available from: https://www.who.int/health-topics/antimicrobial-resistance.

4. Levin-Reisman, I., et al., Antibiotic tolerance facilitates the evolution of resistance. Science, 2017. 355(6327): p. 826-830.

5. Dienemann, C., et al., Crystal structure of the VapBC toxin-antitoxin complex from Shigella flexneri reveals a hetero-octameric DNA-binding assembly. J Mol Biol, 2011. 414(5): p. 713-22.

The cholera causative Vibrio cholerae can adapt rapidly to changing environments via sensory proteins like inner membran regulator ToxR, transducing signals from its periplasmic sensory domain to its cytoplasmic effector domain. ToxR thus activates, co-activates or represses numerous genes in V. cholerae, among them also virulence associated genes. Previously studies suggested that inner membrane protein ToxS plays a crucial role in the activity of ToxR.

The NMR structure of the periplasmic domain of ToxR (ToxRp) reveals the formation of a four stranded β sheet stacked against a long α-helix (Gubensäk et al. 2020). C236, in the middle of the helix forms a disulphide bond with C293 at the C-terminal end. NMR dynamic studies showed that under reducing conditions ToxRp adapts two conformations: one resembling the oxidized form, and a second one revealing strong dynamics proposing an unstructured form. The long C-terminal stretch, including C293, seems to be unstructured and highly flexible under reducing conditions, thereby suggesting an explanation for the increased proteolytic sensitivity of reduced ToxR (ToxRp-red) that was previously reported in V. cholerae (Lembke et al. 2018; Lembke et al. 2020).

By using a combination of NMR, SEC-MALS, and Fluorescence Anisotropy we could identify the formation of a strong heterodimer of the periplasmic domains of inner membrane proteins ToxR and ToxS (ToxRSp) independent on the redox state of ToxRp. Our results reveal that ToxRp binds ToxSp in a 1:1 fashion with a dissociation constant of 11.6nM. Additionally, by monitoring the proteolytic cleavage of ToxRp with NMR we provide a direct evidence of ToxS protective function.

The versatile functions of ToxR propose separate control mechanisms, in order to regulate the activity of ToxR as direct activator, co-activator or repressor. We propose that ToxR activity is mainly controlled by its stability. The reduction of ToxRp cysteines represent one possibility to decrease ToxR stability. This regulation is controlled by periplasmic oxidoreductases DsbA and DsbC in-vivo (Fengler et al. 2012; Lembke et al. 2018; Lembke et al. 2020). The interaction with ToxS represents another possibility to increase ToxR stability by directly protecting ToxRp from proteases DegPS (Lembke et al. 2018; Pennetzdorfer et al. 2019). Binding of ToxS seems to be independent from ToxRp cysteines. Previous work has shown that V. cholerae alkalinizes its surrounding in the late stationary phase, which decreases the interaction between ToxRS and subsequently leads to a loss of function of ToxR due to proteolysis (Almagro-Moreno et al. 2015a; Midgett et al. 2017) Furthermore, our experiments show that binding of ToxSp does not trigger dimerization of ToxRp via disulphide bonds under the applied conditions. Therefore, our data also support the theory that dimerization of ToxR, in order to induce transcription, is activated by the presence of DNA (Midgett et al. 2020; Lembke et al. 2020).

Financial support by the the Austrian Science Fund FWF project T-1239 to Nina Gubensäk is gratefully acknowledged.

Novel FAD-dependent oxidoreductase from a lignocellulose-degrading fungus Chaetomium thermophilum (CtFDO) belongs to glucose-methanol-choline (GMC) superfamily of oxidoreductases which act on the hydroxyl groups of non-activated alcohols, carbohydrates and sterols. GMC superfamily enzymes share a two-domain character, the FAD-binding motif GxGxxG, and usually His-His or His-Asn active-site pair [1]. The crystal structure of CtFDO reveals a unique His-Ser active-site pair a an active-site pocket, which is, compared to known structures of GMC oxidoreductases, unusually large, wide-open, and extended beyond the pyrimidine moiety of FAD. These features of the active-site pocket indicate a different type of substrate than common for GMC oxidoreductases.

A large activity screening with about 1000 compounds including substrates of GMC oxidoreductases showed CtFDO to be inactive toward these compounds. To identify chemical groups of putative substrates and predict the substrates specificity of CtFDO, we utilized the technique of crystallographic fragment screening, which resulted in series of six complexes binding small inorganic and aromatic moieties inside the active-site pocket of CtFDO (Fig. 1). The size of the pocket together with preference for binding of aromatic moieties indicate polyaromatic nature of the putative substrate with molecular weight likely greater than 500 Da.

Figure 1. Crystal structure of CtFDO with color-coded substrate-binding (yellow) and the FAD-binding (blue) domains. The FAD cofactor is shown as sticks with black C atoms and the active-site pocket with salmon surface. (b) Three-dimensional superposition of the active sites of the CtFDO complexes binding four fragments from Frag Xtal Screen (Jena Bioscience) and two other compounds. The active site pocket is displayed as salmon mesh, selected surrounding residues and FAD as sticks with gray C atoms, and the ligands with red, cyan, blank, yellow, green, purple C atoms. Tthe molecular graphics were created using PyMOL (Schrödinger).

[1] Sützl, L. Foley, G., Gillam, E. M. J., Bodén, M., Haltrich, D. (2019). Biotechnol Biofuels 12: 118.

[2] Švecová, L. Østergaard, L. H., Skálová, T., Schnorr, K., Koval’, T., Kolenko, P., Stránský, J., Sedlák, D., Dušková, J., Trundová, M., Hašek, J., Dohnálek, J. (2021). Acta Cryst. D77, 755-775.

The work was supported by the institutional support of IBT CAS, v.v.i. (RVO: 86652036), ERDF (CZ.02.1.01/0.0/0.0/15_003/0000447, CZ.02.1.01/0.0/0.0/16_013/0001776 and CZ.1.05/1.1.00/02.0109), MEYS CR (LM2018127 and CZ.02.1.01/0.0/0.0/16_019/0000778) and by the Grant Agency of the Czech Technical University in Prague (SGS19/189/ OHK4/3T/14).

This poster describes structure-assisted design of inhibitors of human carbonic anhydrase IX (CA IX) based on three-dimensional carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play crucial role in cancer cell proliferation and formation of metastases. The new class of potent and selective CA IX inhibitors combines structural motif of bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn²⁺ atom in the enzyme active site. Detailed structure-activity relationship (SAR) study of a large series containing 50 compounds is corroborated by almost 50 high resolution structures of compounds bound to CA IX active site and the active site of CA II. Structural features of the cluster-containing inhibitors that important for efficient and selective inhibition of CA IX activity were uncovered and used in structure-assisted design. Preclinical evaluation of selected compounds revealed low toxicity, favourable pharmacokinetics and ability to reduce tumour growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/ or for combination therapy in boron neutron capture therapy.

Nearly one-third of the proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches [1, 2]. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria, they are translocated by Na⁺/citrate symporters (CitS) [3, 4]. Interestingly, the presence of a secondary transporter allowing the translocation of divalent metal ion-complexed citrate in Gram-negative bacteria has not been reported till date. In this study, we report the presence of a novel divalent metal ion-complexed citrate uptake system that belongs to the primary active ABC transporter superfamily. For the uptake, the metal ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains (TMDs) for their transport [1, 2]. Since SBPs are involved in maintaining the selectivity and specificity of the substrate(s) and directionality of the transport, they have been reported to be pivotal. This study reports the crystal structures of an Mg²⁺-citrate-binding protein (MctA) from a Gram-negative thermophilic bacteria Thermus thermophilus HB8 in both apo and holo forms at a resolution range of 1.63 to 2.50 Å. Despite binding various divalent metal ions, MctA follows the coordination geometry to bind its physiological metal ion, Mg²⁺. The results also suggest a novel subclassification of cluster D SBPs, known to bind and transport divalent metal ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant proteins of MctA suggests a gating mechanism of ligand entry following an “asymmetric domain movement” of the N-terminal domain (NTD) for substrate binding.

[1] Mandal, S. K., Nayak, S. G. & Kanaujia, S. P. (2021). Int. J. Biol. Macromol.185, 324.

[2] Mandal, S. K., Adhikari, R., Sharma, A., Chandravanshi, M., Gogoi, P. & Kanaujia, S. P (2019). Metallomics. 11, 597.

[3] Kim, J. W., Kim, S., Kim, S., Lee, H., Lee, J. O. & Jin, M. S. (2017). Sci. Rep. 7, 1.

[4] Wohlert, D., Grotzinger, M. J., Kuhlbrandt, W. & Yildiz, O. (2015). Elife. 4, e09375.

Acknowledgements: This work was supported in part by a grant from Department of Biotechnology (DBT), Government of India (Sanction Order No.: BT/PR16065/NER/95/61/2015). SKM acknowledges the Ministry of Human Resource and Development (MHRD), Government of India, for his research scholarship.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids (PLs) between the inner membrane (IM) and outer membrane (OM), thereby maintaining OM asymmetry [1]. The Mla system is a multi-component intermembrane machinery which is composed of three main constituents- an OM MlaA-OmpC/F complex, a free-floating periplasmic protein MlaC and an IM ATP-binding cassette (ABC) transporter complex MlaFEDB [2]. MlaC, which serves as the solute-binding protein (SBP), has been reported to have atypical structural features [3]. However, an in-depth investigation highlighting the peculiarities and the mechanism of ligand binding is still lacking. This study reports, for the first time, the crystal structure of MlaC from Escherichia coli at a resolution of 2.5 Å in a quasi-open state and in complex with a PL. The analysis reveals that MlaC comprises two major domains viz, NTF2-like (D1) and AAA helical-bundle (D2). Each domain can be divided into two subdomains (D1R1, D1R2; D2R1, D2R2) that are arranged in a discontinuous fashion. Further, MlaC would follow a reverse mechanism of binding pocket opening and the subdomains exhibit specific movements that aid in ligand binding and orientation. Based on extensive structural analysis, a novel mechanism of ligand binding is proposed that has not been observed for any known SBP till date. Additionally, the study also highlights the unique ancestries of MlaC and other atypical SBPs that are involved in OM biogenesis. The work firmly establishes MlaC to be a one-of-a-kind transporter protein that plays critical role in maintaining OM asymmetry.

[1] Malinverni, J. C. & Silhavy, T. J. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, 8009. [2] Coudray, N., Isom, G. L., MacRae, M. R., Saiduddin, M. N., Bhabha, G. & Ekiert, D.C. (2020). Elife 9, e62518. [3] Yero, D., Díaz-Lobo, M., Costenaro, L., Conchillo-Solé, O., Mayo, A., Ferrer-Navarro, M., Vilaseca, M., Gibert, I. & Daura, X. (2021). Commun. Biol. 4, 1.

Acknowledgements: This work is supported by Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Grant/Award Number: ECR/2018/000013). AD acknowledges the Ministry of Human Resource and Development (MHRD), Government of India.

Fasciolosis caused by the liver fluke Fasciola hepatica is a worldwide spread parasitic disease of ruminant and an emerging human disease. Cystatin superfamily of cysteine protease inhibitors is composed of intracellular type 1 cystatins (stefins), secreted type 2 cystatins, and multidomain type 2 cystatins. Helminth parasites secrete type 2 cystatins to modulate host immune responses for successful parasitism, except for F. hepatica that lacks type 2 cystatin genes.

This work is focused on F. hepatica type 1 cystatin FhCY2. It was localized to gastroderm and tegument and was surprisingly detected in the excretory/secretory products. We demonstrated that recombinant FhCY2 is a broad-selective inhibitor of host cysteine cathepsins as well as cysteine cathepsins of F. hepatica, suggesting its dual role in the regulation of exogenous and endogenous proteolytic systems. Furthermore, we solved the crystal structure of FhCY2 at 1.6 Å. The structural and phylogenetic analyses revealed that FhCY2 has the sequence and fold of type 1 cystatins but also the signal peptide and disulfides typical for type 2 cystatins, combining all hallmarks in an unprecedented way. We propose that FhCY2 is an evolutionary upgrade of type 1 cystatins for secretion that occurred in F. hepatica (and Fasciolidae family in general) in the absence of type 2 cystatins.

Protein self-association is an extremely common phenomenon in biology. However, light-driven protein homo-oligomerization has so far only been described in a few classes of photoreceptors, most notably plant cryptochromes and phytochromes. The characterization of light-induced protein oligomerization is challenging due to the need of synchronizing sample irradiation with data acquisition. Using a combination of carefully chosen methods, we hereby show that EL222, a bacterial transcription factor belonging to the light-oxygen-voltage (LOV) family, can form clusters in a concentration and power dependent manner. In the dark state, the DNA-binding helix-turn-helix module of EL222 is caged by the adjacent LOV domain. Blue-light excitation of the embedded flavin mononucleotide (FMN) cofactor triggers a cascade of protein conformational changes leading to uncaging of the HTH domain, EL222 dimerization, and interaction with its target DNA. Our time-resolved small-angle neutron scattering (SANS) experiments revealed the kinetics of EL222 assembly into high-order oligomers upon illumination and their subsequent disassembly in the dark. The light-induced changes in EL222 size and shape were found to be fully reversible and the photorecovery rate was in line with the well-known FMN photocycle. Further experiments employing fluorescence correlation (FCS) spectroscopy supported the SANS observations and allowed us to gain more insight into the photoinduced oligomerization kinetics. Analyses of the fluorescence traces and FCS curves pointed to the co-existence of multiple diffusing species in EL222 samples illuminated continuously. Moreover, we identified putative protein-protein interaction interfaces and the role of DNA in the aggregation process. Taken together, our hybrid SANS/FCS approach suggests a plausible mechanism of multimer formation in irradiated EL222.

Transcription is fundamental process for withdrawing genetic information stored in the genome. In eukarya, multi-subunit complexes, known as RNA polymerase II, general transcription initiation factors –i.e. TFIIA, TFIIB, TBP/TFIID, TFIIE, TFIIF, TFIIH–, mediator, and chromatin factors, carry out this reaction. To elucidate the detailed mechanisms of the reaction, their tertiary structural information is indispensable. So far, we determined crystal structures of subunit/domain of TFIID [1,2] and examined molecular evolution of TBP and TFIIB [3,4]. We have also performed large-scale purification of eukaryotic transcription-related complexes for structural analysis [5]. Preliminary cryo-EM analysis showed that these complexes seemed to be disrupted due to the harsh condition during cryo-grid preparation. Further optimization for cryo-grid preparation is required.

March 2018, our institute, KEK, obtained 200kV cryo-EM (Talos Arctica with Falcon3EC) and prepared pipeline for solving high resolution structures of protein complexes. From October 2018, the cryo-EM facility in KEK is open to academic and industrial users for scientific research. Our mission is twofold: to provide cryo-EM machine time for external users, and to assist users in acquiring cryo-EM skills. Until now, we have provided machine time for 36 academic and 15 industrial users. We also have held an initial training for cryo-grid preparation and EPU operation 12 times and a RELION workshop for beginners 3 times. In the last two years, our facility obtained 25 cryo-EM maps whose resolution is higher than 5 angstrom. Here we show two representative results: single particle analyses of 110kDa enzyme at 2.85 angstrom resolution and 860kDa enzymes at 2.24 angstrom resolution. These results suggest that we established a proper protocol for cryo-grid preparation, cryo-EM data collection, and single particle analysis. We would like to keep supporting external users and carry out cryo-EM analysis of transcription-related complexes.

[1] Adachi, N., Senda, M., Natsume, R., Senda, T. & Horikoshi, M. (2008). Crystal structure of Methanococcus jannaschii TATA box-binding protein. Genes Cells 13, 1127-1140.

[2] Akai, Y., Adachi, N., Hayashi, Y., Eitoku, M., Sano, N., Natsume, R., Kudo, N., Tanokura, M., Senda, T. & Horikoshi, M. (2010). Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction. Proc. Natl. Acad. Sci. USA 107, 8153-8158.

[3] Adachi, N., Senda, T. & Horikoshi, M. (2016). Uncovering ancient transcription systems with a novel evolutionary indicator. Sci. Rep. 6, 27922.

[4] Kawakami, E., Adachi, N., Senda, T. & Horikoshi, M. (2017). Leading role of TBP in the Establishment of Complexity in Eukaryotic Transcription Initiation Systems. Cell Rep. 21, 3941-3956.

[5] Adachi, N., Aizawa, K., Kratzer, Y., Saijo, S., Shimizu, N. & Senda, T. (2017). Improved method for soluble expression and rapid purification of yeast TFIIA. Protein Expr. Purif. 133, 50-56.

Keywords: transcription; TFIID; cryo-EM

ABSTRACT

S-adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host E. coli cells, thus inactivating a crucial metabolite involved in plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy ─ it allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle Cryo-EM and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM, but also by interacting with and efficiently inhibiting the host’s methionine S-adenosyltransferase (MAT) ─ the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined together by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency high-amplitude active site-deforming modes. The amplification of uncorrelated motions between active site residues weakens MAT's ability to withhold substrates, explaining the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools.

IMPORTANCE

Self-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy ─ silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes might be a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

The number of high resolution structure determination by cryo-EM single particle analysis (SPA) is growing rapidly. Focusing on maps having better resolution than 3 Å deposited in the Electron Microscopy Data Bank (EMDB), there were 316 depositions in 2019 while it was 82 in 2018. It increases the importance of method developments for accurate determination of atomic coordinates and thus the model validation, where not only the geometric quality but also the fitness to the map is of great importance.

Here we present a new program Servalcat for the refinement and map calculation of cryo-EM SPA structures. Servalcat implements a refinement pipeline using REFMAC5, which uses a dedicated likelihood function for SPA [1]. It takes as inputs unsharpened and unweighted half maps from independent reconstructions. The variance of noise in Fourier coefficients is estimated using the half maps. A weighted and sharpened F_o-F_c map is calculated after the refinement. The Fourier coefficients for the difference maps are derived as expectation values of unknown Fourier coefficients using their posterior distribution given observations and model parameters. Refinement of atomic displacement parameters is crucial for calculation of a sensible F_o-F_c map. It was shown to be useful for visualization of weak features like hydrogen atoms and model errors as it is done routinely in crystallography. Although hydrogen densities are weaker than heavier atoms (e.g. C, N, O), they are stronger than in the electron density maps produced by X-ray crystallography, and some hydrogen atoms are even visible at ~1.8 Å.

About half of the EMDB-deposited SPA maps have non-C1 point group symmetry. If the map has been symmetrised during reconstruction then all downstream programs should be aware of it and the atomic structure model must follow the symmetry. A user can give an asymmetric unit model and a point group symbol to Servalcat for refinement. The NCS constraint function in REFMAC5 was updated to consider non-bonded interactions and ADP similarity restraints between symmetry copies. The MTRIX records in the PDB format and _struct_ncs_oper in the mmCIF format are used to encode the symmetry information. Currently, there are only few asymmetric unit model depositions to the PDB except viruses. We think that refining and depositing asymmetric unit models with annotations of symmetry will be a common practice in future.

We are also developing a new program EMDA, for cryo-EM map and model manipulation with the main focus on validation. EMDA offers several metrics for map validation including FSC combined with mask correction by high resolution noise substitution [2], local correlation using half maps, optimal alignment between maps and magnification scaling using maximum-likelihood method. Also, EMDA includes metrics for map-model validation such as local correlation between map-and-model, which can be used to investigate the quality of the map-to-model fit.

Both EMDA [3] and Servalcat [4] are freely available under an open source licence. They are also available within the CCP-EM package.

[1] Murshudov (2016). Methods in Enzymology 579, 277-305.
[2] Scheres and Chen (2012). Nature Methods 9, 853-854
[3] EMDA https://emda.readthedocs.io
[4] Servalcat https://github.com/keitaroyam/servalcat

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from T. elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. In bacteria, the β-clamp protein forms a homodimer, whereas in eukaryotes or euryarchaeotes, proliferating cell nuclear antigen (PCNA) proteins form homotrimers. However, PCNA from Aeropyrum pernix (ApPCNA), a crenarchaeote species, forms a heterotrimer. The actual structure of ApPCNA-mediated sliding clamps and the mechanism by which they slide along DNA is unknown. Previously, we have analysed the crystal structure of ApPCNA1 from the APE_0162 gene^[1], ApPCNA2 from the APE_0441.1, and ApPCNA3 from the APE_2182 genes^[2]. The present study aimed to analyse the crystal, solution structure and cryo-electron microscopy (cryo-EM) of the heterotrimeric ring of ApPCNA, examine its interaction with DNA and other proteins, and elucidate the mechanism of PCNA function.

Each ApPCNA molecule, which constitutes a heterotrimer, was expressed using the Escherichia coli expression system. The proteins were purified using heat treatment, ammonium sulfate precipitation, and column chromatography. The purified proteins were crystallized using the vapor-diffusion method and the crystals were analysed by X-ray diffraction. To verify the ring shape of ApPCNA2 in solution, the solution structure was analysed using size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). A mixture of ApPCNA1-2-3 and ApPCNA2-3 were analysed by SEC-multi-angle light scattering for the presence of a complex, and the solution structure was analysed by SEC-SAXS. The mixture was analysed by cryo-EM, after purified with gel filtration chromatography.

The solution structure of the ApPCNA1-2-3 complex is similar to shape of the British Isles islands. ApPCNA2 and ApPCNA3 interact in a similar manner as the PCNA rings of other organisms; however, ApPCNA1 is located such that it did not form a perfect ring-shaped structure. The scattering curves of the complex and those of the model edited trimeric ring are almost similar with minor differences. The solution structure of ApPCNA2-3 complex was similar to shape of a naan. This particle contains four subunits rather than trimer. The electron density from cryo-EM forms hexagon.

The solution structure was not trimeric ring, containing ApPCNA1-2-3. The N-terminus of ApPCNA1 is approximately 10 residues longer than that of ApPCNA2 and ApPCNA3. This could be why the tripartite complex is not ring shaped. Moreover, Met16 is present downstream of the N-terminal of ApPCNA1. In the future, the effect of N-terminus deletion and binding of the DNA duplex on ApPCNA1 structure should be evaluated. The solution structure of ApPCNA2-3 complex was not trimeric ring too. In crystal structure of ApPCNA3, the C-terminus interacts between adjacent subunits, probably PIP-Box binding site. This interaction may cause ApPCNA2-3 Complex dose not form ring shape. Generally, PCNA rings that consists of homotrimer have 3-fold symmetry, comprise six edges from concave edge between subunits and flat edge that formed PIP-Box binding site. This hexagonal electron density suggests ApPCNA1-2-3 forms trimeric ring in cryo-EM structure. Interestingly, one of the three edges is completely separated. The Fitting model containing ApPCNA1-2-3 hetero subunits suggests, the long N-terminus of ApPCNA1 cause this separated edge.

Heat shock protein 100 (HSP100) family members serve as ATPases associated with diverse cellular activities (AAA+) chaperones. HSP100s possess three enzymatic activities: ATPase, refoldase and disaggregase/holdase. With these activities, HSP100s play key roles in cellular stress responses and protein homeostasis. The functional oligomeric state of HSP100 is mostly modulated by their hexameric/dodecameric quaternary structures as exemplified by studies on ClpB and ClpC. However, little is known about non-hexameric/dodecameric HSP100 chaperones. We found that ClpL from Streptococcus pneumoniae forms tetradecamers in solution. Here we report the structural characterizations of the tetradecameric ClpL to reveal key residues responsible for the tetradecameric assembly. Cryo-EM structure of ClpL reveals a striking tetradecameric arrangement where two heptameric rings are connected by vertical middle domains. Non-conserved residues, Q321 and R670, are crucial in the heptameric ring assembly of ClpL while hydrophobic F350 contributes to the interface among the middle domains. Site-directed mutagenesis analysis supported the differential roles of the aforementioned residues. Mutations in Q321 and R670 abrogated ATPase and refolase activities, supporting that these residues are critical in the integrity of the heptameric ring arrangement. Mutations in hydrophobic residues of the middle domain deteriorated refoldase and disaggregase/holdase activities. Solution structures of ClpL derived from small-angle X-ray scattering (SAXS) data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric HSP100 chaperone structures. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric HSP100 chaperones.

Interest in cryoelectron microscopy (cryoEM) is growing rapidly as technical advances in electron detectors and optics dramatically improve imaging capabilities and sample throughput. The federal government, via the NIH, has established multiple national centers that provide user access to this technology. Efficient use of these centers requires improved tools and methods for sample management. Building on experience gained in the high-throughput revolution in cryocrystallography, systems for the advanced storage, transport, and tracking of cryo-EM samples are being developed. We report on our current and planned developments in this area.

In recent years serious attention has been focused on various applications of nanodiamonds obtained by detonation synthesis of carbon-containing explosives (detonation nanodiamonds - DNDs) [1]. DNDs find applications in biomedicine, development of novel liquid heat carriers and magnetic liquids. Thus, study of their rheological properties in various liquid media is of great importance [1]. Two rheological features of hydrosols have been recently discovered: the sharp increase in viscosity and the phase sol-gel transition at a relatively low filler concentration [2]. Such behaviour has been explained in the frame of percolation model based on network formation from the chains of faceted DND particles due to electrostatic interaction of facets [3]. The comprehension of DNDs structural organisation in various media is crucial for practical applications and it would be important to obtain a direct experimental evidence of DND chains existence in sol and network formation at the sol-gel transition. Such direct observation has been successfully performed by Cryo-Electron Tomography (Cyo-ET): the processes of bonding and agglomeration of the DND particles were observed, and the 3D spatial distribution and quantitative analysis, including fractal analysis, were accomplished on the basis of Cyo-ET data. The obtained results explain the rheological properties of the DNDs hydrosols.

The study was focused on two types of DNDs hydrosols with positive and negative electrokinetic potential (ζ-potential) in the concentration range from 1.0 to 7.0 wt%. Cryo-ET study was performed on Titan Krios 60-300 TEM/STEM (FEI, USA) at acceleration voltage of 300 kV.

The 3D models of positive and negative ζ-potential DNDs were obtained and the 3D spatial distribution of DNDs was revealed [4]. The data demonstrate the formation of extended fractal structures and chains of individual faceted DND particles. The skeletonization procedure was applied in order to evaluate the bonding of objects and percolation. It was observed that DNDs with positive ζ-potential form a percolation network. However, such network was not observed in DNDs hydrosols with negative ζ-potential. This explains the differences in the rheological behavior at low concentrations of samples with different sign of ζ-potential. The 2D and 3D fractal dimensions were calculated for the mass-fractal and fractal surface from the Cryo-ET data. The fractal dimensions are in good correlation with the small angle X-ray scattering (SAXS) data for the fractal dimension of a DNDs cluster. Moreover, the distances between DND particles were estimated after the 3D reconstruction of the specimen volume. Interparticle distance distributions showed, that the secondary maximum position qualitatively matches the interplanar distance between fractals at 1 wt% contain of DND particles in hydrosol measured by SAXS.

The results of 3D reconstruction and analysis based on Cryo-ET data allowed to explain observed features of the rheological behavior associated with DNDs agglomeration and chain formation.

[1] Shvidchenko, A.V., Eidelman, E.D., Vul', A.Ya. et al. (2019). Adv. Colloid Interface Sci. 268, 64.

[2] Vul', A.Ya., Eidelman, E.D., Aleksenskiy, A.E. et al. (2017). Carbon. 114, 242.

[3] Kuznetsov, N.M., Belousov, S.I., Stolyarova, D.Yu. et al. (2018). Diam. Relat. Mater. 83, 141.

[4] Kuznetsov, N.M., Belousov, S.I., Bakirov, A.V. et al. (2020). Carbon. 161, 486.

The detonation nanodiamonds hydrosols were kindly provided by Prof. Vul' A.Ya. and coworkers from Laboratory Physics for Cluster Structures of Ioffe Institute. This work was partially supported by Russian Foundation for Basic Researches, project 18-29-19117 mk.

Crystallization of active drug molecules in the presence of various biologically acceptable molecules (acids, bases and amino acids) with the aim of forming a new drug composite has gained immense importance in the last couple of decades [1]. This targeted co-crystallization is exercised to enhance the physical and biological properties of the APIs in the pharmaceutical industry [2]. Low aqueous solubility, poor intrinsic dissolution rate (IDR) and moisture sensitivity of the parent various drug molecules have triggered us to investigate the possibility of formation of novel salts of existing drugs for enhanced physical properties and better biological activity [3]. Levofloxacin (LFX) [4], a broad-spectrum antibiotic suffers from low aqueous solubility and poor IDR. Co-crystallization of LFX with natural organic acids has yielded novel crystalline salts of LFX. Characterization by FTIR, PXRD and DSC confirmed the formation of the new phases and single crystal X-ray diffraction data confirmed the formation of salts as well. Enhanced solubility and IDR of the resultant salts motivated us to conduct in-vitro and in-vivo biological study on selected salts. Minimum inhibitory concentration (MIC) of LFX and salts were determined in E. coli and S. typhimurium. Inhibitory concentration IC₅₀ was determined in S. typhimurium infected Caco-2 cells. Pharmacokinetics parameters and biodistribution study (in heart, liver, kidney and brain) of LFX and selected novel salts using 1 CBM peroral Balb/c mice model was conducted. These salts have shown significant improvement in MIC and IC₅₀ then LFX. So, these salts are more potent then pure drug. These salts are more water soluble and we have seen this effect in the pharmacokinetic parameters like absorbance, plasma half life time, T_max, C_max, bioavailability, elimination rate constant and clearance of salts. Significant results of our study will be presented.

[1] Brittain, H. (2012). Cryst. Growth Des. 12, 5823

[2] Jones, W., Motherwell, W. D. S., Trask, A. V. (2006). MRS Bull. 31, 875.

[3] Joshi, M., Choudhury, A. R. (2018). ACS Omega. 3, 2406.

[4] Davis, R.; Bryson, H. M. (1994) Drugs, 47, 677.

Pharmaceutical cocrystallization has been an active field of research in the last couple of decades. A large number of research groups have been working in this area and have contributed significantly in the development of new materials derived from known active drug molecules. A number of reviews [1-3] have summarised the contributions of all the major research groups working in the area. In the last decade, our group has been involved in the development of salts and cocrystals of a library of drug molecules, which pose various challenges in formulations due to their poor aqueous solubility and low dissolution rates or high moisture sensitivity. Our experiments on fluconazole, voriconazole, valproic acid, enrofloxacin, lamivudine, amoxapine, levofloxacin, ofloxacin, etc has demonstrated a range of exciting results.

Our efforts in forming cocrystals of fluconazole (antifungal agent) with various monobasic and dibasic acids have resulted into a series of new polymorphs of the parent drug instead of formation of salts or cocrystals [4]. These results highlighted the importance of possible intermolecular interactions between the drug and the conformer in solution. In contrary, voriconazole (antifungal agent) resulted into a cocrystal with a dibasic acid. Valproic acid (mood stabilizing agent), which is liquid at room temperature, is available in the market as a sodium salt, which is highly moisture sensitive and dissolves in moisture soon. Our experiments with valproic acid resulted into stable crystalline salts with a few organic bases. Enrofloxacin, a well-known broad spectrum antibiotic, which also suffers from poor aqueous solubility, has resulted into a series of highly water soluble salts using solvent drop assisted grinding experiments [5].

Amoxapin, a tricyclic antidepressant, also suffers from poor solubility and dissolution rate. Our experiments have resulted into a few stable and highly water soluble salts of amoxapine [6]. Ofloxacin and Levofloxacin were also targeted for the formation of salts with pharmaceutically acceptable organic acids. Novel salts of these drugs were tested for their biological activity and based on enhancement in activity; salts of Levofloxacin were further tested for their activity in animal model as well. Our results indicated that the novel salts of Levofloxacin were more potent than the existing formulations.

Significant results achieve in last 10 years on these drugs from our laboratory will be highlighted in the presentation with special emphasis on their synthesis, characterization, physical and biological (both in-vivo and in-vitro) property studies of novel salts developed in our laboratory.

References:

[1] Brittain, H. (2012). Cryst. Growth Des. 12, 1046.

[2] Brittain, H. (2012). Cryst. Growth Des. 12, 5823.

[3] Brittain, H. (2013). J. Pharm. Sci. 102, 311.

[4] Karanam, M., Dev, S. & Choudhury, A. R. (2012). Cryst. Growth Des. 12, 240.

[5] Karanam, M., & Choudhury, A. R. (2013). Cryst. Growth Des. 13, 1626.

[6] Joshi, M. & Choudhury, A. R. (2018). ACS Omega. 3, 2406.

Most pharmaceuticals are administered in solid form, therefore extensive research and development efforts are invested to explore the phase diagram of APIs and solid-state formulations. The elucidation of the crystal structure of crystalline forms is the key tool for assessing their applicability, as it allows the rationalization of their relevant physicochemical properties, bioavailability, manufacturing, stability etc. Nonconventional crystallization methods[1] enable to discover new solid forms including polymorphs, cocrystals and solvates, allowing to see even known APIs under a new light. However, these methods can easily lead to nanocrystalline products, which hamper structural elucidation.

3D Electron diffraction (3D ED)[2] has recently emerged as a powerful tool for the discovery of new crystalline forms of pharmaceutical compounds,[3-5] as it allows to bypass many of the common bottlenecks of this process and of the established characterization methods based on x-ray diffraction. Small crystal size, mixture of phases, small product quantities are very frequent obstacles to the structural characterization of APIs that can be easily overcome by 3D ED methods.

Here we showcase how our electron diffractometer, fully dedicated to 3D ED experiments, represents a revolutionary innovation for the discovery of new crystal forms of APIs. Our recent results of representative case studies dealing with challenging pharmaceutical compounds will demonstrate the performances of a dedicated device and how it can meet the growing needs of the crystallographic and pharmaceutical community.

[1] Potticary, J., Hall, C., Hamilton, V., McCabe, J. F. & Hall, S. R. (2020). Cryst. Growth Des. 20, 2877.

[2] Gemmi, M., Mugnaioli, E., Gorelik, T. E., Kolb, U., Palatinus, L., Boullay, P., Hovmöller, S. & Abrahams, J. P. (2019). ACS Cent. Sci. 5, 1315.

[3] Andrusenko, I., Potticary, J., Hall, S. R. & Gemmi, M. (2020). Acta Cryst. B76, 1036.

[4] Hamilton, V., Andrusenko, I., Potticary, J., Hall, C., Stenner, R., Mugnaioli, E., Lanza, A. E., Gemmi, M. & Hall, S. R. (2020) Cryst. Growth Des. 20, 4731.

[5] Andrusenko, I., Hamilton, V., Mugnaioli E., Lanza, A., Hall, C., Potticary, J., Hall, S. R. & Gemmi, M. (2019). Angew. Chem. Int. Ed. 58, 10919.

We gratefully acknowledge Dr. Mauro Gemmi and Dr. Iryna Andrusenko (Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy) for fruitful discussions.

Thalidomide (TD) was first developed as a safe sedative hypnotic in 1958 and was prescribed for pregnant women to prevent morning sickness. However, TD was reported to cause the tragic side effect in 1960s: Babies born to mothers who took TD had small limb or malfunctioning organs. Nevertheless, TD has attracted much attention as an indispensable pharmaceutical compound because of its effectiveness in treating intractable diseases. Meanwhile, Blaschke et al. reported that only (S)-isomer of TD caused its teratogenicity [1]. This report has influenced pharmaceutical researchers, and then the importance of studies on chirality of drugs have been recognized. However, subsequent studies revealed facile racemization of TD in the aqueous solution [2, 3], which led us to discuss the interpretation for Blaschke’s report. Since then, many researchers in various scientific fields have been interested in the mechanism of teratogenicity of TD.

In 2010, the molecular target of TD was identified Cereblon (CRBN) [4], which forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 [5]. The structural and biochemical studies on the R- snd S-enantiomers of TD binding to CRBN has been reported, which has revealed that the S-enantiomer exhibits ca.10-fold stronger binding to CRBN compared to the R-enantiomer [6].

Furthermore, the important and significant study on TD have been reported [7, 8], which can demonstrate Blaschke’s report. Meanwhile, experimental results on TD itself in the crystalline state were not sufficiently done. Especially, optical properties in TD crystals were not revealed because of the difficulty in growing the good single crystal. We have succeeded in growing thin single crystals of TD with sublimation methods and studying optical properties in them using the Generalized High Accuracy Universal Polarimeter abbreviated as G-HAUP, which enables us to measure simultaneously linear birefringence (LB), linear dichroism (LD), circular birefringence (CB; optical activity), and circular dichroism (CD) in anisotropic materials [9-11].

We will show wavelength dependencies of anisotropic optical properties; LB, LD, CB, and CD, in TD single crystals.

[1] Blaschke, G., Kraft, HP., Fickentscher, K., Kohler, F. (1979). Arzneimittel-Forschung, 29, 1640-1642.
[2] Nishimura K., Hashimoto, Y., Iwasaki, S. (1994). Chem. Pharm. Bull., 42, 1157-1159.
[3] Knoche, B., Blaschke, G. (1994). J. Chromatogr. A, 666, 235-240.
[4] Ito, T., Ando, H., Suzuki, T., Ogura, T., Hotta, K., Imamura, Y., Yamaguchi, Y., Handa, H. (2010). Science, 327, 1345-1350.
[5] Fischer, E. S., et al. (2014). Nature, 512, 49-53.
[6] Mori, T., Ito, T., Liu, S., Ando, H., Sakamoto, S., Yamaguchi, Y., Tokunaga, E., Shibata, N., Handa, H., Hakoshima, T. (2018). Sci. Rep., 8, 1294.
[7] Maeno, M., Tokunaga, E., Yamamoto T., Suzuki, T., Ogino, Y., Ito, E., Shiro, M., Asahi, T., Shibata, N. (2014). Chem. Sci., 6(2), 1043-1048.
[8] Tokunaga, E., Yamamoto, T., Ito, E., Shibata, N. (2018). Sci. Rep., 8, 17131.
[9] Kobayashi, J., Asahi, T., Sakurai, M., Takahashi, M., Okubo, K., Enomoto. Y. (1996). Phys. Rev. B, 53, 11784-11795.
[10] Tanaka, M., Nakamura, N., Koshima, H., Asahi, T. (2012). J. Phys. Appl. Phys., 45, 175303-175311.
[11] Nakagawa, K., Asahi, T. (2019). Sci. Rep., 9, 18453.

The transition metals, specifically biometals such as Co, Cu, Zn and Ni, are being the target of numerous scientific studies from different branches of chemistry, medicine and pharmacology. These metals are well known to form bonds and interactions with biomolecules. Also, are often responsible for the biological function of biomolecules in the body, are immersed in many biochemical processes essential for life. In addition, these metal cations have a great tendency to form coordination compounds with numerous types of ligands. Into the medical-pharmacological field is the medicinal inorganic chemistry that explores binding agents with therapeutic properties linked to metal cations and their multiple applications.^1,2

this work focuses on the synthesis, characterization and structural study of complexes based on metformin and transition metals as Co(II), Cu(II), Ni(II) and Zn(II), in order to propose new therapeutic alternatives, by taking advantage of the characteristics of current drugs in synergy with the activity of metallic cations.³

Ritonavir is a drug of the protease inhibitor class, marketed by Abbvie™ - as of 1996 under the name of Norvir^® - for the treatment of adult and pediatric patients infected with HIV. Lopinavir is a protease inhibitor drug used in combination with ritonavir in therapy and prevention of HIV infection. Both drugs display polymorphism, which may lead to severe commercial implications for pharmaceutical manufacturing [1]. One way to overcome such problems is the development of amorphous formulations like Kaletra commercial medicine. In this work, we use a ball-mill system and an agate mortar and pestle to obtain individual amorphous ritonavir and lopinavir samples as well as their mixtures. First of all, we identify and characterize the crystal forms present in the raw samples of lopinavir and ritonavir, as well as quantify the mass concentrations of each crystal phase using X-ray powder diffraction and the Rietveld method. Ritonavir tends to recrystallize after some time. On the other hand, the mixture of an already amorphous lopinavir sample with ritonavir seems to facilitate its amorphization. The ball-mill processing of ritonavir and lopinavir together results in the production of unexpected crystalline forms of ritonavir. Pair distribution function (PDF) analysis shows the mixed samples reveal a higher r-dependence of ritonavir up to 2 Å (first two peaks). At the same time, the lopinavir dependence tends to increase for a higher-r signal.

Modern instrumentation and processing techniques enable high-quality 3D structure analysis – including absolute structure determination – often in less than an hour, faster and more comprehensively than many spectroscopic methods can even start to achieve. However, large numbers of small or highly flexible organic molecules remain intractable to even the most sophisticated crystallization methods. Our new set of chemical chaperones for co-crystallization, developed by the University of Stuttgart^[1] offers a new alternative to other methods, such as the crystal-sponge approach^[2] and can significantly increase the probability of successful crystallization and provide faster access to the absolute 3D structure of an organic analyte:

• The chaperone method is fast and easy to use
• Structures in hours rather than weeks
• Small quantities of analyte required
• Excellent quality crystals
• Sample screen of 52 organic compounds
  • Diffraction-quality crystals in 88% of cases
  • High resolution X-ray structures in 77% of cases
  • The chaperone compounds are highly stable
  • 100% analyte occupancy in the crystal guarantees reliable determination of the absolute configuration

We will discuss and demonstrate the features in detail along the diastereomers of Limonene including a demonstration of the crystal growth.

^[1] Angew. Chem. Int. Ed. 2020, 59, 15875–15879.

^[2] Patent pending.

Molecular structure determination is beneficial for the development of medicines, aroma chemicals, and agrochemicals. Single crystal X-ray diffraction (SC-XRD) analysis is the most powerful technique for molecular structure determination. However, SC-XRD analysis requires good quality crystals.

In fact, the biggest hurdle for SC-XRD analysis is crystallization. Crystallization trials require a large amount of highly purified target compounds. Moreover, good quality crystals for SC-XRD analysis might not be obtained despite performing tedious and time-consuming trials. In this case, we have to abandon the direct structure determination by SC-XRD. As one way to address this situation, Fujita et al. have reported the crystalline sponge method (CS method) for the structure determination of small molecules [1]. However, as with other analysis techniques, the CS method has some limitations.

The CS method utilizes the MOF as the pre-crystallized 'container' for the analytes. The 'container' equips flexible features to fit various analytes and must have enough space to accommodate the wide variety of the molecules. In the latter mean, MOF is a large structure object with three-dimensional networks; thus, the spaces to accommodate are to have limitations in principle.

To overcome the above difficulty, we came to an idea of 'molecular grabber', utilizing protein that has a multisite binding pocket to bind a variety of types of molecules, and having characteristics on easy to crystallize, and resulted crystal gives high-resolution spots.

In this presentation, we will indicate the initial results of the "Molecular Grabber" method, utilizing RamR as the molecular grabber (Fig. 1).

[1] Inokuma, Y., Yoshioka, S., Ariyoshi, J., Arita, T., Hirota, Y., Takada, K., Matsunaga, S., Rissanen, K. & Fujita, M. (2013). Nature 495, 461-467. [2] Matsumoto, T., Nakashima, R., Yamano, A. & Nishino, K. (2019). Biochem. Biophys. Res. Commun. 518, 402-408.

Mefenamic acid (MefA) is one of the non-steroidal anti-inflammatory drugs (NSAID) commonly used in the treatment of mild to moderate pain. Some of its metal derivatives have shown greater pharmacological activity than mefenamic acid, in addition to fewer side effects of the acid in the digestive tract. With this in mind, it was considered of interest to prepare simple metal complexes of MefA. The reaction of Co(CH₃COO)₂·4H₂O and sodium mefenamate (prepared from NaOH and MefA) in water at ambient conditions, produced a purple precipitate which was filtered and washed with MeOH:water. FT-IR indicated this was a Co-Mefenamic acid derivative. After a solubility study, the product was recrystallized from N,N-dimethylformamide (DMF) by slow evaporation at room temperature. Very small and thin pink needles were obtained after approximately 4 weeks. These crystals were characterized by ATR-IR spectroscopy and single crystal X-ray diffraction. This material crystallizes in a monoclinic P2₁/c (No. 14) unit cell with an unusually large volume: a = 15.9550(2) Å, b = 33.5553(11) Å, c = 31.6703(10) Å, β = 90.898(2)°, V = 16953.4(8) ų, Z = 4.

Structure determination and refinement showed a complex structure based on a cluster of nine octahedrally coordinated crystallographically independent cobalt atoms, eight mefenamate ligands, six bridging hydroxyl groups, six DMF molecules, one MeOH, three water molecules, and two carbonato moieties at the core of the cluster. NaCO₃ was identified as an impurity in the NaOH used to prepare the Na-Mefenamate reagent. The mefenamate ligands coordinate in a bridging bidentate mode and exhibit intramolecular N—H···O hydrogen bonds. Intermolecular H-bonds occur between carboxylate oxygens, water, DMF, and MeOH molecules. Additional π···π and C—H···π interactions are important in stabilizing the structure. It is worth noting that only three related compounds were found in a search of the CSD. Of them, only one compound (Refcode HAJGIJ) has a similar Co₉ core [1].

The coupling of nitroxide and tertiary phosphane moieties offer unique opportunities in synthesis and catalysis as well as in biological effects. Surprisingly, no crystal structure report is found in CSD [1] for tertiary phosphane substituted pyrroline nitroxide. We first reported such structure [2] and synthesis of 3-(diphenylphosphino)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radical, 1, Fig.1., left. Structural data clearly indicate the expected nitroxide radical. Analysis of supramolecular structure gave interesting results (Fig. 1., right). A simple derivative had shown better antiproliferative effect on MDA-MB-231 and MCF-7 human breast cancer lines than MITO-CP indicating the potential of the compound for use in cancer therapy [3]. Moreover, inclusion of phosphane and nitroxide moiety into the same ligand suggest versatile homogeneous catalytic activity by both metal center and ligand assisted mechanisms and they may serve as organocatalysts, too. Further studies to explore these potentials are under way in our laboratories.

Figure 1. ORTEP view of 1 with selected bond length (Å) and angle (°) data (left) and packing diagram (right) showing voids with a small probe.

[1] Groom, C.R., Bruno, I.J., Lightfoot, M.P., Ward, S.C. (2016), Acta Cryst. B72, 171-179 .DOI: 10.1107/S2052520616003954

[2] Isbera, M., Bognár, B. Gallyas, F., Bényei, A. Jekő, J. & Kálai, T. (2021). Molecules, in press. [3] Andreidesz, K.,Szabó, A., Kovács, D., Kőszegi, B.,Vantus, V.B., Isbera, M., Kálai, T., Bognár, Z., Kovács, K., Gallyas, F. (2021). International Journal of Molecular Sciences, submitted for publication

Keywords: IUCr2020; abstracts; template (use Keywords style, Arial 9pt, bold, and separate keywords by semicolons)

This research was funded by the Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the 2020-4.1.1.-TKP2020

Gefitinib is a chemotherapeutic drug used in the treatment of breast and lung cancer. It is belonging to Biopharmaceutical Classification System II (BCS II) as it is highly permeable and poor soluble drug. The bioavailability of gefitinib is significantly affected by low aqueous solubility of the drug. To overcome this issue, we prepared co-crystals of gefitinib with resorcinol using dry grinding and solution growth method. Crystal structures of newly synthesized co-crystals were analysed by single crystal X-ray diffractometer, and it reveals that gefitinib formed 1:1:1 co-crystal with resorcinol and water. Quantitative analysis intermolecular interactions were studied using Hirschfeld surface and 2D fingerprint plot analysis. Weak molecular interactions like H∙∙∙C, H∙∙∙O, Cl∙∙∙H, F∙∙∙H and N∙∙∙H interactions play a significant role in gefitinib co-crystal formation.

Cetirizine dihydrochloride and levocetirizine are antihistamines of second-generation that block histamine receptors H₁, are widely used to treat allergic symptoms. These compounds belong to the class of antihistamines piperazine type and like other second-generation antihistamines, are considered non-sedating [1]. The crystal structure of cetirizine dihydrochloride has been solved and refined using X-ray powder diffraction data and optimized using Density Functional Theory (DFT) techniques. The cetirizine dihydrochloride Fig. 1, crystallized in a monoclinic system and space group P2₁/n (Nº 14) with parameters a=13,6663(3) Å, b=7,0978(7) Å, c=23,8311(1) Å, β=102, 488(3)°, V=2251,06 ų and Z=4. On the other hand, the levocetirizine dihydrochloride Fig. 1, crystallized in a monoclinic system and space group P2₁ (Nº 4) with parameters a=13,5450(7) Å, b=7,0719(9) Å, c=24,0527(2) Å, β=98, 159(3)°, V=2280,65 ų and Z=2. In both crystalline structures there are multiple hydrogen bonds intra and inter molecular, π-interactions and hydrogen-π interactions. The molecular packing and crystal energy are dominated by Van der Waals attractions according to Hirshfeld surfaces. Finally, the crystal structure was optimized with DFT and all non-H bond distances and angles were subjected to restraints, based on a Mercury Mogul Geometry Check of each molecule.

A search in the Cambridge Structural Database (CSD) [2] confirmed the absence of reports for the crystal structure of cetirizine dihydrochloride and levocetirizine. However, there are several reports of cetirizine dihydrochloride and levocetirizine in the PDF-4/Organics database [3] contains four entries PDF 00-058-1973, 00-058-1974 and 00-058-1975, corresponding to unindexed patterns about cetirizine dihydrochloride, dextrocetirizine dihydrochloride and levocetirizine dihydrochloride, respectively; PDF 00-066-1627 corresponding an experimental pattern for cetirizine dihydrochloride, according to this report, it crystallizes in a monoclinic cell with parameters a=24.1256(7) Å, b=7.07588(7) Å, c=13.5196(4) Å β=98.0028(28)° and V=2285.45 ų in space group P2₁/n (Nº14).

Solvates are ubiquitous in pharmaceutical crystalline solids [1]. Such solvates of a given drug molecule can exhibit different physicochemical properties such as solubility, thermal stability, and mechanical strength [2]. Hence, it is of extreme importance to understand the role of solvent molecules in determining the mechanical properties of the solvates via the nanoindentation technique, especially in view of the manufacturing of pharmaceutical tablets as well as the recent understanding of structure‐property correlations in molecular crystals [3].

The current investigation reveals the role of guest lattice solvents in tailoring nanomechanical responses (hardness and elastic modulus) in different crystalline forms of two pharmaceutically active compounds, in particular with respect to the supramolecular structure and energetics of interaction topology of molecules. The nanomechanical responses of two crystalline phases of a dihydropyrimidine analogue are similar irrespective of the presence (or absence) of the guest dichloromethane lattice solvent [4]. In contrast, the mechanical properties of two differently solvated forms (acetonitrile and DMSO) of the second related compound are anisotropic (Fig. 1). The structural features of all crystalline forms demonstrate that depending on the presence of specific guest solvent molecule and interaction topology of host-guest intermolecular interactions along with their relative orientations in the crystal lattice, majorly decide their nanomechanical properties (hardness and elastic modulus).

Figure 1. Guest solvent-dependence of nanomechanical properties in pharmaceutical crystalline solids.

[1] Griesser, U. J. (2006). The importance of solvates. In Polymorphism in the Pharmaceutical Industry (Ed.: R. Hilfiker), Wiley-VCH: Germany, pp. 211–257.

[2] Brittain, H. G. (2009). Polymorphism in Pharmaceutical Solids, Informa Health-care, NewYork.

[3] Wang, C. & Sun, C. C. (2020). CrystEngComm 22, 1149.

[4] Bhandary, S., Rani, G., Mangalampalli, S. R. N. K., Rao, G. B. D., Ramamurty, U and Chopra, D. (2019). Chem. Asian J. 14, 607.

Keywords: pharmaceutical solvates; nanoindentation; mechanical properties and interaction topology

Azobenzene molecules exhibit reversible light-triggered changes in geometrical structure (cis/trans isomerism) and nanoscale mechanical properties. For this reason, they have have been already widely used in materials science to build photo-mechanical responsive systems, incorporated in electronic switchable devices, used for graphene slicing, optical switching and data storage. We have been exploring further the photoresponsive nature of newly designed azobenzene derivatives and exploiting the potential of these smart materials for the generation of novel low-cost relevant molecular machines for biotechnology enabling the control of production/degradation of amyloid-based biomaterials. To this end, we have carefully functionalized azobenzene molecules by properties-by-design approach supported by the state-of-the-art in silico molecular design techniques as well as structure determination by X-ray crystallography. According to our Thioflavin-T Assay and NMR experimental results, the custom-designed azobenzene switches interact with the amyloid assemblies and intercalate between their strands. Stimulation with light, by inducing conformational change of azobenzene molecules, puts mechanical stress on the amyloid strands, eventually dissociating them and, in turn liquidizing the amyloid fibrils. Our in vitro studies of the designed azobenzene derivatives indicate no evidence of their cytotoxicity. Hence, it should be possible, in general, to use them for photo-control amyloid degradation in living systems, which constitutes a big encouragement for designing new azobenzene derivatives for biomedical applications, for example novel therapies against severe infections caused by amyloid biofilm forming bacteria or amyloid associated neurogenerative conditions such as Alzheimer’s disease.

Thiazolidinone derivatives play crucial roles in anticancer (breast cancer, lung cancer and leukaemia) drug discovery process.¹ Especially, 5-arylidene-2-aminothiazolidinones are found to show antimitotic activities against MCF7 breast cancer cells.² However, bioactive molecules are known to undergo polymorphic modifications under certain conditions and polymorphs often exhibit distinct physicochemical and biopharmaceutical properties.³ Indeed, polymorphism has been the critical issue in drug development process.⁴ Therefore, systematic characterization of polymorphism in bioactive molecules is highly essential. Here, we report the discovery of polymorphism on 5-arylidene-2-aminothiazolidinones derivatives and their systematic characterizations via single-crystal X-ray diffraction, thermal analyses and spectroscopic analysis. The estimation of energies in terms of interaction energies, lattice energies and energy frameworks bring out the energetic stabilities of the polymorphs. Solubility, dissolution rate and phase stability experiments confirm that the thermodynamically most stable form exist with least solubility and dissolution rate. Further, we investigated the extent of inhibition imposed by our library of polymorphs on the proliferation of MCF7 breast cancer cell lines and also the extent of their binding to the target enzyme (g-enolase). Our preliminary cellular assay suggest that in general specific polymorphic forms are indeed more potent than the corresponding bulk form. The g-enolase binding assay also demonstrated a trend similar to that of the phenotypic screening against MCF7. Furthermore, the binding affinity of the polymorphs with g-enolase as estimated via molecular dynamic simulations is in well agreement with the binding assay results. The aforementioned experiments in general emphasized the importance of polymorphism in improving the biological potency of the molecules. We believe that the studies of this kind would help screening potent drug molecules in pharmaceutical industries.

Last decades there is an ongoing interest in materials with reversible temperature-triggered single-crystal-to-single-crystal phase transitions (SCSC-PT) because of the huge fundamental attraction to this phenomenon as well as the promising practical applications of SCSC-PT as different kinds of switches (e.g. to switch dielectric properties) and memory-devices [1-2].

On the other hand, hydrogen bonds are considered as the powerful and flexible synthon in the field of crystal engineering particularly to obtain materials possessing phase transitions. Hence, keeping it in mind we have obtained a series of complexes with the general formula [CaZn₂(piv)₆(L)₂], where piv – pivalic acid anion, L – MeOH, EtOH, THF.

Structures of all coordination complexes were unambiguously determined using single-crystal X-Ray Diffraction (XRD) analysis. All compounds form supramolecular 1D-chains either via hydrogen bonds (in the case of L = MeOH and EtOH) or via CH…O-contacts (in the case of L = THF). The XRD analysis revealed that complex [CaZn₂(piv)₆(EtOH)₂] has interesting temperature-induced isosymmetric reversible phase transition (about 250K on cooling and about 280K on heating) with large hysteresis loop where ethanol groups’ rotation being as a main driving force of the process. As a result of rearrangement of hydrogen bonds network, molecules of the low temperature phase become considerably bent with the angle Zn…Ca…Zn 166.45° compared to completely linear molecules at room temperature.

On the contrary, complex [CaZn₂(piv)₆(MeOH)₂] does not show phase transitions in the available temperature range 100K – 293K. The geometry of its molecules is similar to the low temperature phase of [CaZn₂(piv)₆(EtOH)₂], but the angle Zn…Ca…Zn is 167.30° at 150K. Noteworthy, even though the XRD analysis did reveal rotation of one of the MeOH groups, the cell parameters are roughly the same between structures obtained at RT and 150K. Apparently, because of much less steric hindrance and spatial degrees of freedom of MeOH groups, the temperature of SCSC-PT lies much higher beyond 293K to be revealed.

[1] Ge, J.-Zh, Fu, X.-Q., Hang, T., Ye, Q., & Xiong R.-G., (2010). Cryst. Growth Des., 10, 8, 3632.

[2] Kendin, M. & Tsymbarenko, D. (2020). Cryst. Growth Des., 20, 5, 3316.

Ferroelectric effects are observed in wide variety of materials such as composite ceramics, solids, polymers, crystals and liquid. Various inorganic compounds, such as, barium titanate (BaTiO3), potassium dihydrogen phosphate (KDP), Lithium niobate (LiNbO3), lead zirconate titanate (PZT) etc are well known for decades. On the other hand, organic ferroelectrics in recent times have attracted considerable attention in the material science due to their potential applications as ferroelectric random access memories (FeRAM), non volatile random access memories (NVRAMs) and dynamic random access memories (DRAMs) etc. However, single component organic compounds that show multifunctional properties have hardly investigated and very few reports are available in the literature.

In the present investigation, we report the crystal structures of two N-Benzylideneaniline analogues (BOA and HBOA) and analyse their hydrogen bonding interactions, second harmonic generation efficiency, ferroelectric and dielectric properties. Both the molecules are crystallized in a non-centrosymmetric with monoclinic space group P2₁ and stabilized by strong intermolecular interactions through O-H…O and N-H…O hydrogen bonds. SHG activity of BOA and HBOA also confirms their non-centrosymmetric nature which is prerequisite characteristics for ferroelectric materials. SHG efficiency was found to be 68mv and 140mv for BOA and HBOA respectively with respect to KDP (75mv, standard). Further large thermal hysteresis was observed from the DSC scan of BOA and HBOA at the range of 100 ºC and 87 ºC respectively. Furthermore both the compounds have shown significant reversible mechanofluorochromic (MFC) behaviour upon grinding and fuming. The reversible MFC behaviour was confirmed by cognate techniques like powder X-ray diffraction (PXRD), thermal analysis and fluorescence studies. Both the systems were further investigated to demonstrate the structure-property relationship for realizing their ferroelectric behaviour by PUND (positive up and negative down). Interestingly, both the molecules are fascinating ferroelectric behaviour at high electric field and tolerate upto 181-182 kV/cm without any breakdown (upto instrument limitation). The origin of ferroelectricity has been observed due to the proton transfer in both the systems (Shown in Fig. 1). The dielectric loss-factor was measured in a wide range of frequency at room temperature. High dielectric constant and low loss of energy indicate that the materials can be good candidate for energy storage application. The combination of findings provides a potential perspective for designing new organic multifunctional materials for the applications in light displaying and memory devices.

Lapachol (systematic name: 2-hydroxi-3-(3-methyl-but-2-enyl)-[1,4]naphtoquinone) is a 1,4-naphtoquinone, representative of a large class of natural compounds that exhibit a wide range of biological effects, acting as antibacterial, antifungal, antiparasitic, antiviral, antileishmanial and anticancer, among others [1]. The crystal structures of two polymorphs of lapachol LAPA I (triclinic P ̅1, a= 5.960(1), b= 9.569(2), c= 10.679(2) Å, α= 96.82(2), β= 98.32(2) and γ= 90.32(2) °) and LAPA II (monoclinic P2₁/c, a= 6.035(1), b= 9.427(2), c= 20.918(5) Å and β=98.27(2) °) were determined by Larsen et al. at 105 K [2]. In the course of our research on novel synthetic approaches and crystal structure determination of known and new derivatives of lapachol [3], crystals of a new lapachol polymorph were obtained. Lapachol (Fig. 1a) was obtained from an extract of Handroanthus heptaphyllus (Vell.) Mattos (pink trumpet tree or lapacho negro) and purified by recrystallization by slow evaporation from an EtOH:Et₂O mixture to obtain yellow crystalline plates that correspond, mostly, to LAPA II (monoclinic P2₁/c, a= 6.0550(2), b= 9.5769(2), c= 21.2391(5) Å and β= 98.2910(10) °, T=293 K). However, when lapachol was recrystallized from ethyl acetate, large yellow plates also containing lapachol molecules but arranged in a new crystalline form, were obtained. This new polymorph, LAPA III, also belongs to the monoclinic system, with space group P2₁/c and cell parameters a= 9.6134(5), b= 6.0119(3), c= 21.5464(11) Å and β= 96.760(2) ° at 293 K.

Lapachol molecules show the same conformation in the three structures and crystallize forming H-bonded dimers between nearby OH and ketone groups from two molecules related by an inversion center. In the three structures the dimers stack together in a staggered manner to form double layers of molecules with the butenyl moiety pointing outward. These layers seem to be very stable, since the C face of the unit cell is identical in the three compounds with a≈ 6.0(1), b≈ 9.6(1) Å and γ≈ 90.0(4) °. The difference between them lies in the way successive layers stack to form the crystal. In the triclinic LAPA I all the layers stack in identical orientation with alternating butenyl residues from the bottom and top layers (Fig. 1b). The stacking in LAPA II, however, occurs with the next layer rotated 180° and shifted b/2 respect to an axis parallel to b between the layers (z= ½ of the triclinic cell or ¼ of the monoclinic ones). This rotation axis transforms b in the monoclinic axis of the cell and also introduces a c-glide plane normal to b. This makes the two parallel layers different along c, so LAPA II exhibits a doubled c-axis. Additionally to a, b and γ, β (≈ 98.3°) is also conserved between both structures because the addition of symmetry requires no change in the relative position of the layers along b. LAPA III shows exactly the same relation with LAPA I except that the addition of symmetry occurs in the triclinic a-axis, so the conventional monoclinic unit cell given above shows a and b axes exchanged respect to LAPA I. Again, the addition of a 2₁ axis and a c-glide makes a-axis the unique one, and produces a unit cell that conserves a, b, γ (as LAPA II) and the triclinic α angle (≈ 96.7°) transformed to b, and a doubled c-axis (Fig. 1c). Considering this, it is only the stacking of lapachol double layers what determines the polymorphic form. Hirschfeld surface analysis and intermolecular interaction energy calculations were performed to identify the main interlayer interactions to better explain the differences between the crystal structures of the three lapachol polymorphs.

En este trabajo sintetizamos, caracterizamos y estudiamos por difracción de rayos X monocristal y polvo, dispersión Raman y cálculos de mecánica cuántica, la estructura de una serie de bifenilos sustituidos en posiciones 3,3´, 4,4´- con una variedad de grupos R conectados al núcleo de bifenilo a través de átomos de oxígeno (R: metilo, acetilo, hexilo). [1,2] La serie de seis miembros se dividió en dos grupos con diferencias notables en la conformación molecular, así como en los puntos de fusión (a saber ., en el estado sólido tres miembros son estrictamente planos y presentan un pf significativamente más bajo, mientras que los tres restantes están muy retorcidos, con un pf mayor). Así, el objetivo del trabajo es conocer si alguno de los fragmentos moleculares intervinientes ejerce alguna influencia decisiva sobre la planaridad molecular así como sobre la estabilidad térmica de los compuestos.

La conformación molecular, en particular el ángulo de torsión entre anillos aromáticos, se ha estudiado extensamente tanto en estado sólido como líquido. Los resultados muestran que los tres compuestos que aparecen como rigurosamente planos en el sólido (según lo evaluado por Difracción de rayos X de monocristal) presentan en lugar de una conformación retorcida en la masa fundida (como se describe mediante experimentos Raman y / o cálculos de mecánica cuántica). El tema sólido vs fundido sugiere fuertemente que las razones se encuentran en las restricciones del empaque, aunque no es fácil encontrar una explicación sencilla: en algunos casos (como aquellos con las cadenas de sustitución más cortas) las interacciones combinadas no unidas pueden ser señalados específicamente como responsables de los efectos, mientras que en algunos otros (como en los más largos), pueden ser más sutiles y difusos, no atribuible directamente a interacciones específicas. [2] Finalmente, se sugiere una “regla empírica” para el diseño de bifenilos con diferente conformación molecular, basada en la selección del OR utilizado. [2,3]

[1] Zelcer, A., Cecchi, F., Alborés, P., Guillon, D., Heinrich, B. y Cukiernik, FD (2013). Liq.Cryst. , 40 , 1121. [2] Vadra, N., Suarez, SA, Slep, LD, Manzano, VE, Halac, EB, Baggio, RF & Cukiernik, FD (2020). Acta Cryst. B, cumbre. [3] Grineva, OV (2009). J. Struct. Chem. 50 , 727.

Agradecemos el apoyo económico de la Universidad de Buenos Aires (becas UBACyT 20020130100776BA y20020170100512BA) y CONICET (número de beca PIP20110101035 y becas a NV (PhD) y VEM (postdoc)).

Meloxicam (MLX), a widely used anti-inflammatory drug, can crystallize as one of four neat polymorphic forms and one hydrated form, as reported in the original MLX patent [1]. However, only its commercially available form I and hydrated form IV have their crystal structure determined and were obtained in their pure forms by other researchers, with forms II, III and V remaining elusive. Recently, having found an admixture of a small amount of form III of MLX in one of the commercially available drug formulations, Freitas et al. attempted to obtain pure form III by repeating crystallization procedures described in the patent, but all trials remained unsuccessful [2].

In this contribution we describe our efforts to obtain and characterize three elusive polymorphs of MLX using Crystal Structure Prediction (CSP) calculations and various crystallization approaches. Each of the three solid forms required a different approach, among which were meticulous and numerous repetitions of examples described in the patent, application of various crystallization additives and gel phase crystallization. Crystal Structure Prediction calculations were exploited in two different ways. First, they were used to indicate the prevalent hydrogen bonding patterns found in energetically favourable crystal structures of MLX in order to rationalize possible crystallization routes. It was found, for example, that form II, identified on the calculated CSP landscape by comparison of the experimental and theoretical powder X-Ray diffractograms, display different hydrogen bonding (HB) pattern than commercially available form I (Figure 1). This led us to perform crystallization experiments using nitrogen-containing heterocycles as additives [3] and gel phase crystallization with a hope to re-direct the crystallization route from form I to the three elusive polymorphs.

The second way of exploiting CSP calculations was to characterize the obtained polymorphs of MLX, in combination with NMR crystallography approach. All of the elusive polymorphs of MLX crystallized as microcrystalline powders, which excluded a possibility of characterizing them with single-crystal X-Ray diffraction. Instead, solid-state NMR under very fast magic angle spinning conditions and PXRD diffraction were used to guide the calculations, identify crystal structures of the elusive polymorphs on the CSP landscape, as well as to validate the calculated structures. The presented results are a step towards understanding a fine interplay between the crystal structure and the method of crystallization of elusive polymorphs.

[1] Coppi, L., Sanmarti, M. B. & Clavo, M. C. (2003). US patent 2003/0109701 A1. [2] Freitas, J. T. J., Santos Viana, O. M. M., Bonfilio, R. Doriguetto, A. C. & de Araujo, M. B. (2017). Eur. J. Pharm. Sci. 109, 347. [3] Thomas, L. H., Wales, C. & Wilson, C. C. (2016). Chem. Commun. 52, 7372.

This work was financially supported by Polish National Science Center (UMO-2018/31/D/ST4/01995). PL-GRID is is gratefully acknowledged for providing computational resources.

Active research of some organic compounds, which are actual or promising drugs, has led in the last decade to the production of a large number of their new polymorphic modifications [1]. To date, examples of compounds with up to 12 structurally studied modifications are known. From the point of view of crystal chemistry, such highly polymorphic systems are of great interest for studying the relationship between structure and properties, since their composition is fixed. At the same time, in the presence of suitable methods of analysis, one can try to establish the influence of the slightest changes in the geometry of molecules (conformation) or their packing on the properties of the final substances. Such changes inevitably affect interatomic interactions in the crystal structures of these compounds.

To study both chemical bonds and van der Waals interactions, classical crystal chemistry uses a comparison of interatomic distances with different tabulated systems of radii. However, practice shows that this approach is insufficient for such complex objects as highly polymorphic compounds. Another method for studying noncovalent interactions, the Hirshfeld surface method, implies a visual one-by-one comparison of compounds and takes into account only about 95% of the crystal volume, which also leads to many limitations.

In the course of this long-term project, we consider highly polymorphic systems within the framework of the stereoatomic model of crystal structures [2]. The advantage of this model is that it takes into account all possible interactions (chemical bonds and intra- and intermolecular noncovalent contacts) from uniform positions in all 100% of the crystal volume. In addition, this method allows computer programming and, as a consequence, automatic analysis in large data samples, which is especially important given the volume of already accumulated and progressively increasing information about crystal structures.

Many known highly polymorphic systems have already been successfully analysed using the stereoatomic model of crystal structures. This list includes such champions in terms of the number of structurally studied modifications as flufenamic acid (8 modifications) [3], aripiprazole (9 modifications) [4], galunisertib (10 modifications) [5], etc. For all compounds, various parameters of all types of interatomic interactions in crystal structures were calculated, which can be used for subsequent searches for all kinds of relationships. One of the main results, which was first shown using the example of these compounds, not qualitatively, but quantitatively, is that each polymorphic modification corresponds to a unique set of noncovalent contacts, what, in our opinion, is one of the reasons for the existence of polymorphism.

In the course of this project, additional tools for crystal chemical analysis were developed. For example, one of them is the k-Φ criterion, which makes it possible to unambiguously and objectively identify conformational polymorphs [6]. The (RF, d) distributions were introduced in assistance to the k-Φ criterion to get an idea of the relationship between the ranks of faces and the corresponding interatomic distances [5]. Also, a method was developed to visualize changes in noncovalent interactions when changing the geometry of molecules [7].

As a result, all the work done brings us closer to understanding how the differences in the energetics of polymorphic modifications can be explained from the standpoint of individual noncovalent interactions - this is the question posed by Professor J. Bernstein in his book in 2002 [1].

The study was funded by a grant of the Russian Science Foundation (project number 20-73-10250).

[1] Bernstein, J. (2002). Polymorphism in Molecular Crystals. Oxford University Press: New York. [2] Serezhkin, V. N. (2007). Some Features of Stereochemistry of U(VI). In Structural Chemistry of Inorganic Actinide Compounds, edited by S. Krivovichev, P. Burns & I. Tananaev, pp. 31–65. Elsevier Science. [3] Serezhkin, V. N. & Savchenkov, A. V. (2015). Cryst. Growth Des. 15, 2878. [4] Serezhkin, V. N. & Savchenkov, A. V. (2020). Cryst. Growth Des. 20, 1997. [5] Serezhkin, V. N. & Savchenkov, A. V. (2021). CrystEngComm. 23, 562. [6] Serezhkin, V. N. & Serezhkina, L. B. (2012). Crystallogr. Rep. 57, 33. [7] Savchenkov, A. V. & Serezhkin, V. N. (2018). Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 74, 137.

Since the early 2000’s several free-distribution and commercial computer programs have advanced crystal structure determination from powders (SDPD). The free-distribution software WinPSSP [1] consists of a graphical user interface for an essentially unmodified version of PSSP [2], which uses a reconstructed X-ray powder diffraction pattern (a pseudo-pattern) made of correlated peak intensities calculated from the results of a Le Bail fit [3] (FWHM, peak positions, multiplicity factors and squared structure-factor amplitudes). The simulated annealing algorithm [4] is used to locate the asymmetric unit (provided in Cartesian coordinates) in the unit cell, using a space group symmetry candidate, a cost function (quantitatively evaluating the agreement between the diffracted intensity in the pseudo-pattern and that calculated from a large number of trial models), once a set of structural parameters (fragment positions, Eulerian angles and torsional angles) have been defined by the user. The crystal and molecular structures of more than fifty small-molecule organics have been solved with PSSP and WinPSSP. After Rietveld refinement (typically done with GSAS [5]), those have been published in peer-reviewed journals, or the atomic coordinates have been submitted together with reference powder diffraction patterns to the Powder Diffraction File database [6]. In most cases, synchrotron X-ray powder diffraction from capillary transmission geometry has been used.

This work analyses the distribution of various indicators among the structures solved, such as internal, external and total degrees of freedom, space group symmetries, Z, Z’, unit cell volumes, number of atoms in the asymmetric unit (with and without hydrogens) and number of fragments to independently locate. The uses of solid-state characterization techniques providing information that enabled or confirmed SDPD (thermogravimetry, differential scanning calorimetry, NMR, optical microscopy and DFT calculations) is also highlighted. SDPD common impediments (e.g., preferred orientation from laboratory powder diffraction data) will be indicated.

Even though simple structures can be solved in the order of minutes using WinPSSP [2], due to the approximately exponential increase of the number of trial models required while the number of structural parameters increases [2], a challenge for the current search algorithm is to simultaneously and correctly locate many crystallographically independent fragments (typically three or more) using a reasonable number of trial models, comparable with that used in SDPD with small to moderate size asymmetric units. WinPSSP [2] facilitates the relocation of fragments incorrectly positioned in initial runs (as deemed by the user, considering data from varied solid-state characterization techniques and likely chemical bonding), and examples of this capability toward solving large structures will be discussed.

Another work goal is to attract undergraduate students to SDPD, whenever possible reducing the black box use of crystallography software. Guided SDPD examples have been presented in two workshops and will be used in a yearly SDPD workshop at Old Dominion University.

[1] Pagola, S., Polymeros, A. & Kourkoumelis, N. (2017). J. Appl. Cryst. 50, 293-303. [2] Pagola, S. & Stephens, P. W. (2010). J. Appl. Cryst. 43, 370-376. [3] Le Bail, A. (2005). Powder Diffraction 20, 316-326. [4] Kirkpatrick, S., Gellat, C. D. & Vecchi, M. P. (1983). Science 220, 671-680.

[5] Larson, A. C. & Von Dreele, R. B. (2004). General Structure Analysis System (GSAS). Los Alamos National Laboratory Report 86-748.

[6] Gates-Rector, S. & Blanton, T. (2019). Powder Diffraction 34, 352-360.

Keywords: powder diffraction; direct-space methods; simulated annealing; SDPD; crystal structure solution from powders

Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. SP gratefully acknowledges partial funding from the International Centre for Diffraction Data (ICDD) through GIA 08-04 and workshop sponsorship, and partial funding and other support from the Chemistry & Biochemistry Department at Old Dominion University.

Coordination compounds are obtained by the reaction between Lewis acids (metal) and Lewis bases (ligand). According to Pearson’s acid-base theory, also known as HSAB theory, the stability of the compound depends on the hardness of the acid and the base and their affinity. Hard bases tend to react with hard acids and soft bases prefer to react with soft acids. By just varying the hardness of the metal, the ligand or the solvent it is possible to substantially change the structure of the complex, giving a plethora of possibilities for the synthesis of different coordination compounds. This phenomenon was evidenced in coordination compounds of Cu (II) and Zn (II), where changing the ligand from 3,5-dinitrobenzoate to pyrazole derivatives affects the number of metallic centers present after crystallization [1]. Interested in these results we decided to study the effect of the recrystallization solvent on the coordination sphere of the complex [Co(3,5-dinitrobenzoate-O,O’)₂]. Depending on the donor capabilities of the solvent, the complex undergoes a change on its coordination sphere, changing from a trinuclear Co (II) complex when the solvent is a soft base to mononuclear Co (II) complex when the solvent is a hard base. These structural changes are of great interest because materials and molecules that include cobalt in their structure have several spin states, which gives it interesting magnetic properties [2].

Due to its anti-adherent and lubricating properties, magnesium stearate is the most used additive in pharmaceutical products [1]. Most products contain a few percent of magnesium stearate. The compound exists in several hydrated states, and the state of hydration has important consequences for the lubricating functionality [2]. Yet, none of the crystalline phases has been structurally determined despite the extensive use of this compound in pharmaceutical and other industries for over several decades [3]. The reason for that might be that commercially available samples are usually not pure; they contain a significant amount of magnesium palmitate – a stearate homologue differing by two CH₂ groups. Furthermore, it seems to be problematic to obtain large enough single crystals suitable for a conventional X-ray diffraction experiment due to extremely low solubility of this material. We were able to synthesize highly pure magnesium stearate and obtain micrometre size single crystals suitable for a microdiffraction experiment at an X-ray synchrotron facility. The structure of magnesium stearate trihydrate could be determined. This is the first structurally characterized magnesium stearate hydrate phase. Our work could facilitate structure determination of other magnesium stearate phases as well those of magnesium palmitate from PXRD data. The structural data would be immensely useful to understand lubricating and other structure-determined properties of this extensively used material.

[1] Lakio, S., Vajna, B., Farkas, I., Salokangas, H., Marosi, G., Ylirussi, J. (2013). AAPS PharmSciTech. 14, 435-444.

[2] Ertel, K. D., Carstensen, J. T. (1988). J. Pharm. Sci. 77, 625-629.

[3] Bracconi, P., Andrès, C., Ndiaye, A. (2003). Int. J. Pharm. 262, 109-124.

Water molecules are omnipresent in nature and are a key part of many life processes. Due its ability of hydrogen binding water molecule plays an important role in the packing of small molecule crystal structures. Over the past years, the structure of a water molecule has been intensively studied. [1] The experimental values for a free water molecule in the gas phase are the bond angle (H–O–H) of 104.52 ± 0.05° and the bond (O–H) length of 0.9572 ± 0.0003 Å. [2] Neutron diffraction experiments of liquid water showed that the bond angle increases to 106.1 ± 1.8° and the bond length increases to 0.970 ± 0.005 Å. [3] Most of the bond angles in structures of ice have values close to a tetrahedral angle. However, in some of the ice structures, the bond angles and bond lengths remarkably deviates. Calculations based on the spectroscopic potential energy surface showed the equilibrium structure of a water molecule with the bond angle of 104.501 ± 0.005° and the bond length of 0.95785 ± 0.00005 Å. [4] In this study, [5] we performed an analysis of non-coordinated water containing structures archived in Cambridge Structural Database (CSD) as well as ab-initio calculations on a range of bond angles and bond lengths of water molecule. The results of the analysis of crystal structures solved by neutron as well as by X-ray diffraction analysis showed a large discrepancy of both the bond angle and bond length values. Namely, the ranges of the bond angle and the average bond lengths of neutron solved structures having R factor ≤ 0.05 are from 100.74° to 113.92° and from 0.91 Å to 0.99 Å respectively. The corresponding range of the bond angle of X-ray solved structures is from 13.27° to 180.00°. High level ab initio calculations predicted a possibility for energetically low-cost (±1 kcal mol–1) changes of both the bond angle and bond lengths in a wide range, from 96.4° to 112.8° (Fig. 1) and from 0.930 A to 0.989 A (Fig. 1), respectively. Consequently, it would lead to at least 15% of X-ray solved structures that contain questionable water molecule geometries.

In recent years, quantum crystallography, which combines X-ray crystal structure analysis and quantum chemistry, has attracted attention. Attempts have been made to reproduce wave function from electron density obtained from experiments and to refine the crystal structure using computational chemistry [1,2]. However, these attempts require a high degree of expertise and such programs are not widely used. The purpose of this study is to discuss electronic states of azo-Schiff base metal complexes based on quantum chemical calculations and to verify whether quantum crystallography can be performed easily by using conventional programs.

The samples investigated were two Schiff base metal complexes having azobenzene moiety (new Cu of trans-[CuN₂O₂] and known Mn of cis-[CuN₂O₂X₂]) (Fig. 1) studied on photochemical behavior [3]. Experimental electron density was drawn using a PLATON program. DFT calculation was carried out with a Gaussian09, and electron density analysis and bond order analysis were also performed. Additionally, a CRYSTAL EXPLORER program was used for Hirschfeld surface analysis. Experimental and calculated electron density maps exhibited good agreement (Fig. 2) and gave additional information such as bond strength only with the aid of DFT.

[1] Grabowsky, S.; Genoni, A.; Burgide, H.-B. Chem. Sci., 2017, 8, 4159–4176.

[2] Genoni, A.; Bučinský, L. Claiser, N.; Contreras-García, J.; Dittrich, B.; Dominiak, P. M.; Espinosa, E.; Gatti, C.; Giannozzi, P.; Gillet, J.-M.; Jayatilaka, D.; Macchi, P.; Madsen, A. Ø.; Massa, L.; Matta, C. F.; Merz Jr., K. M.; Nakashima, P. N. H.; Ott, H.; Ryde, U.; Schwarz, K.; Sierka, M.; Grabowsky, S. Chem. Eur. J., 2018, 24, 10881-10905.

[3] Takiguchi, Y.; Onami, Y.; Haraguchi, T.; Akitsu, T. Molecules, 2021, 26, 551 (12 pages).

Hydrogen bonds are of greate importance for understanding of different processes in chemistry, crystalography and biology. [1] Properties of hydrogen bonds were subject of numerous experimental and theoretical studies. [1] Specially interesting case represent hydrogen bonds involving transition metal complexes since coordination of ligands can have significant influence on electrostatic potentials of coordinated molecules. [2] Here we present detailed analysis of crystalographic data combined with quantum chemical calculations of very strong hydrogen bonds between water and acetylacetonate ligand of different transition metal complexes.

Cambridge Structural Database (CSD) was searched for all structures containing O-H/O interactions between water molecule and acetylacetonato ligands of transition metal complexes. Geometrical parameters extracted from crystall structures were analyzed and compared with quantum chemical calculations performed on model systems. The O-H/O interactions were studied on model systems containing water and neutral or charged square-planar complexes of Ir, Rh, Pd, and Pt. The strongest interactions were found in charged model systems and these results are in agreement with the predominant electrostatic nature of hydrogen bond (-16.54 kcal/mol). However, suprisingly strong O-H/O interactions were identified also in neutral model systems. The calculated energies of these interactions are -7.98 and -8.22 kcal/mol in [M(acac)(en)]/H₂O (M = Ir(I), Rh(I)) model system, respectively.

Using geometrical cristeria for hydrogen bonds 82 structures with 220 O-H/O contacts involving water molecule and coordinated acetylacetonato fragment were found. All extracted structures were statistically analyzed and results of analysis were in agreement with the results of quantum chemical calculations on model systems.

Although the metal is not directly involved in hydrogen bonding, the results of theoretical studies show that the nature of metal atom has significant influence on the strength of hydrogen bonds of ligands.

[1] T. Steiner, Angew. Chem. Int. Ed., 2002, 48-76.

[2] G. V. Janjić, M. D. Milosavljević, D. Ž. Veljković, S. D. Zarić, Phys. Chem. Chem. Phys, 2017, 8657-8660.

Molecular crystals with structural phase transitions are expected as novel materials for actuators and sensors. The design of crystals with phase transitions has been challenged by experimental and theoretical approaches. However, the former approach requires huge time and cost for experiments, and the latter requires high computational accuracy due to the high degree of freedom of molecular conformation. In recent years, the inductive approach to obtain knowledge from known data has been attracting attention as materials informatics. In this study, the intermolecular interactions in the (S)-N-3,5-di-tert-butylsalicylidene-1-(1-naphthyl)ethylamine (enol-(S)-1) crystals were analyzed, and we found new insights on structural phase transitions.

The enol-(S)-1 crystal has three crystal phases, namely α (<-80°C), β (-80~40°C), and γ (<40°C) phases, which are reversible through single-crystal-to-single-crystal phase transition depending on temperature change[1]. First, the lattice energies were calculated at each temperature point of the enol-(S)-1 crystals to elucidate the whole strength of intermolecular interactions. The lattice energies showed a discontinuous temperature dependence according to the phase transition, with a slight decrease at the phase transition from α to β phase and then an increase with rising temperature (Fig.1a). Then, the analysis of intermolecular interactions by Hirshfeld surfaces and 2D fingerprint plots was performed to reveal which interaction has contribution on the phase transitions. The Hirshfeld analysis uncovered that the proportion of π···π interactions decreased while the proportion of CH···π interactions increased at the phase transition from α to β phase. In addition, while the proportion of π···π interaction did not change, the proportion of CH···π interaction decreased in the β phase as the temperature rose but increased slightly after the transition from the β to γ phase. As to interaction energies, it was shown that the intermolecular interaction of CH···π stabilized at the transitions from α to β phase and from β to γ phase (Fig.1b). CH···π interactions had the unique temperature dependence compared to other main interactions of π···π and CH···π interactions. These results clarify the contribution of CH···π interaction to the stability of the high-temperature crystal phases and may provide new insights for designing crystals with phase transitions.

The development of new procedures to refine experimental diffraction data lead to an increased number of individual software packages to perform these analyses. They may require setup of specific input files, learning of configuration files, and sometimes result in file types unique to each package, which can make comparisons, changes between methods, and the overall workflow time consuming and only available to a trained specialist. NoSpherA2 – Non-Spherical-Atoms-in-Olex2 [1] – provides a possibility to interface any type and source of atomic form factors to the refinement engine olex2.refine [2], itself based on the cctbx [3].

This interface makes it possible to combine any level of sophistication in the calculation of the form factors, ranging from tabulated spherical atoms to tailor-made form factors from quantum mechanical calculations with the established refinement engine. Restraints, constraints, disorder modeling, solvent masking, and intuitive handling using the well-known Graphical-User-Interface of Olex2 [4] are the main advantages, which in combination with easy selection of options, automatic completion of CIFs, and no required manual handling of input files make the treatment of diffraction data using non-spherical models easier than ever.

While NoSpherA2 provides a variety of possibilities and generally better results of refinements (compare Figure 1), some questions about handling of various structures arise: Is it possible to mix-and-match different approaches? How to handle network compounds like MOFs and inorganic Salts? If we describe the non-spherical density distribution of atoms, what information possibly left in the data might need improved treatment? What resolution is required to use NoSpherA2?

The benefits, common practices, and strategies to tackle problems when using NoSpherA2 will be presented with examples and the philosophy of the development: Making the best-suited model available for the refinement task to obtain the best possible results without the need of individual file-conversion, in-depth training or specialized extra software.

[1] Kleemiss, F., Dolomanov, O. V., Bodensteiner, M., Peyerimhoff, N., Midgley, L., Bourhis, L. J., Genoni, A., Malaspina, L. A., Jayatilaka, D., Spencer, J. L., White, F., Grundkötter-Stock, B., Steinhauer, S., Lentz, D., Puschmann, H., Grabowsky, S. (2021), Chem. Sci. 12, 1675-1692. [2] Bourhis, L. J., Dolomanov, O. V., Gildea, R. J., Howard, J. A. K., Puschmann, H. (2015), Acta Cryst. A71, 59-75. [3] Grosse-Kunstleve, R.W., Sauter, N.K., Moriarty, N. W., Adams, P. D. (2002), J. Appl. Cryst. 35, 126-136. [4] Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K., Puschmann, H. (2009), J. Appl. Cryst. 42, 339-341.

Analysis of charge density distribution is a powerful method to recover information about non-covalent interactions in crystals and such important physical quantities as lattice energies. These quantities allow evaluation of the mutual stability of polymorphs and comparison of the strength of supramolecular associates in solids that is valuable for crystal engineering. Moreover, in the case of compounds that serve as active pharmaceutical ingredients (API) the energies of individual intermolecular interactions and lattice energies can be associated with the energies of ligand-receptor binding.

Herein we present the results of experimental charge density studies, quantum chemical calculations and Voronoi partitioning for several APIs (abiraterone acetate [1], bicalutamide [2] and lamivudine) used in common practice to treat tumors and HIV infection. As result the energies of individual interatomic interactions were evaluated for single crystals of API and simplified models describing ligand-receptor interaction constructed using PDB data as starting points. The characterization of intermolecular interactions was carried out with a variety of theoretical approaches including deformation electron density, QTAIM theory, NCI method, molecular electrostatic potential and solid bond angles (Fig. 1). The data on intermolecular interaction obtained for single crystals and models of ligand-receptor binding demonstrated the similarity of lattice energy values with those for the energies of interactions between API and receptor despite of conformational changes.

The analysis of experimental electron density and Voronoi analysis of intermolecular bonding of compounds studied was financially supported by the Russian Science Foundation (project 20-13-00241).

There are already several examples of successful potential antineoplasic copper complexes which have completed pre-clinical trials, such as Casiopeína-III-ia [1] with copper(II) and HydroCuP® [2] with copper(I), that show high selectivity towards cancer cells. This work presents some of the structure-property relationships that we have determined in different potential antitumor agents during the last years in the laboratory. We focused particularly in intermolecular interactions in the crystal structure and their relationship with the lipophilicity of the compounds and the extent and mode of interaction with DNA, considered one of the main target biomolecule for complexes with planar aromatic ligands.

In the study of copper(I) ternary complexes with diimine and triphenylphosphine ligands there is a relationship between the percentage of polar interactions seen in the 2D fingerprint plot derived from Hirshfeld surface analysis and the experimentally determined lipophilicity [3]. Heteroleptic copper(II)-neocuproine complexes were also studied using L-dipeptides as co-ligands to regulate the lipophilicity of the obtained complexes and their ability to interact with DNA (Fig. 1). Studies confirmed that the complex with the most C-C interactions is the only one that interacts with DNA through partial intercalation, whereas the rest interact through groove-binding [4]. We are currently working in complex-model membrane interactions through an interdisciplinary approach that includes the use of full interaction maps to aid the understanding of the experimental and molecular modelling docking results.

This work was supported by: FAPESP, CAPES (Brazil), CSIC and PEDECIBA (Uruguay).

Drug-drug cocrystals involve the combination of two or more active pharmaceutical ingredients (API) with their original chemical characteristics maintained without breaking or forming new covalent bonds, thus ensuring its effectiveness.[1] Its pharmaceutical properties are determined by the polarity of functional groups, the electrostatic potential and the available intermolecular interactions, which in turn are characterized by the three-dimensional crystalline arrangement and governed by its molecular electronic structure.[2] These molecular electron properties and their relationships with the charge density topology can be analysed by experimental and theoretical studies.

In this manner, the experimental charge density analysis of the pharmaceutical drug-drug cocrystal involving the antimetabolite prodrug 5-Fluorocytosine (5-FC) and the tuberculostatic drug Isoniazid (INH), named 5FC-INH, [3] has been carried out based on the Hansen & Coppens aspherical multipolar model refinement,[4] using high resolution X-ray diffraction data at low temperature ((sin(θ_max)/λ=1.15 Å^-1, 150K). The experimental model was compared with those derived from corresponding theoretical calculations for solid-state and gas-phase conditions using density functional theory (DFT) methods at the B3LYP6-311++G** level of theory.[5] The detailed study of the molecular electron density, its corresponding topology and charge distribution were based on the quantum theory of atom in molecules (QTAIM).[6] The charge density distribution and analysis of topological properties revealed that the C—F bond may have a transit closed-shell configuration (Fig. 1). This analysis also allowed to verify the charge delocalization due to resonance-assisted hydrogen bond (RAHB) in the formation of the heterosynthon that stabilizes the crystal packing.[7]

Solvatochromism, thermochromism and photochromism are just a few examples of phenomena involving stimulated colour change. The external factors influencing absorption can be physical and/or chemical and might lead to variations of materials hue as well as cause limitations of light transmission [1]. Those effects find multiple applications in photochromic lenses, as smart self-dimming windows, paints and indicators, thermal papers, visual displays or biochemical probes. It is of particular interest for those processes to be controllable (selective absorption) and reversible. To achieve this goal it is necessary to gain more knowledge on the origin of those effects.

Recently we have studied (pseudo)polymorphs of tyraminium violurate showing both crystallochromic and solvatochromic effects [2]. We have deduced the origin of colour for each of the three phases using a set of quantum crystallography tools. The fluctuations in the electron density within the oxime group of violurate ions (target) was proven to be one of the factors influencing the absorption. This research forced us to think outside the box and formulate more general guidance criteria on how to design new chromic materials based on a common target molecule (chromogen) and changing selectively co-formers.

In this work, we have engineered a series of chromic materials based on violuric acid and its derivatives. We have chosen co-formers e.g. aromatic, aliphatic amines or pyridine derivatives to obtain a group of distinctly coloured products. The obtained crystal phases were further analysed using UV-vis and NMR spectroscopy as well as XRD and hot stage microscopy.

QTAIM analysis [3,4] in combination with H¹ NMR has enabled us to formulate a more general mechanism of colour generation in the violurate family. The possible phase transitions and influence of the temperature on the colour change in the solid-state were examined using hot stage microscopy and PXRD. The obtained results will contribute to a better understanding of chromic effects in the solid-state as well as in solution. Uncovering the origin of colour in one family of chromogens enables us to influence the absorption of a material by means of cocrystallization.

[1] Bamfield, P., Hutchings, M. (2018) Chromic Phenomena: Technological Applications of Colour Chemistry, Royal Society of Chemistry. [2] Gryl, M., Rydz, A., Wojnarska, J., Krawczuk, A., Koziel, M., Seidler, T., Ostrowska, K., Marzec, M. & Stadnicka, K. M. (2019). IUCrJ 6,
226-237. [3] Bader, R. F. W. (2003). Atoms in Molecules: A Quantum Theory, International Series of Monographs on Chemistry, Vol. 22. Oxford: Clarendon Press. [4] AIMAll (Version 19.10.12), Todd A. Keith, TK Gristmill Software, Overland Park KS, USA, 2019 (aim.tkgristmill.com)

This research was supported by National Science Centre Poland, grant number UMO-2018/30/E/ST5/00638

Intermolecular interactions are very crucial point to understand exhaustively in the part of rational drug design. Interestingly, the electronic level information of these intermolecular interactions is not possible to determine without high resolution x-ray diffraction measurements. However, this measurement is more familiar for small molecules and still demand to challenge the protein-ligand complexes. The design of drug with improved physical and chemical properties are major driving forces in the medicinal chemistry. To achieve this, quantum crystallographic approach helps to estimate the stability of interactions obtained from the ligand molecule with their target amino acid residues. Indeed, recent methodology development reports [ref] helped us to study as well as compute intermolecular interaction energies of protein-ligand complexes. Therefore, the present study is mainly focused to determine the different type of interactions between protein and ligand at electronic level. To accomplish this, the desirable protein-ligand complexes like enzyme-drug, enzyme-inhibitor and metal proteins-inhibitor complexes were subjected to QM/MM calculation followed by quantum theory of atoms in molecules (QTAIM) analysis which helps to understand the strength of intermolecular interaction and charge density distribution of protein-ligand complexes and these results compared with already reported experimental results. Electron density, Laplacian of electron density and hydrogen bond dissociation energy of metal interactions are very higher than the other interactions which confirms that the metal coordination is partially covalent bond. Hirshfeld surface analysis along with subsequent fingerprint maps were plotted to quantify the intermolecular contacts between ligand and amino acid residues. Non-Covalent Interaction (NCI) analysis has proved method for the qualitative analysis of hydrogen bonds which plays a crucial role and the accurate NCI energies of these bonds are essential to understand the binding mechanism in the formation drug-receptor complexes. NCI isosurface map of intermolecular interactions of protein-ligand complex clearly visualized the strong and weak interactions. Therefore, the quantum crystallographic based interaction energy calculation is a better alternative to docking score-based modelling. The results will be discussed at the time presentation.

In recent years, Single Molecule Magnets (SMMs) have gained significant attention, primarily due to their potential technological applications in the field of information storage and processing. SMMs are molecules that behaves as small nanomagnets and represents the smallest possible bit that can be used to store binary information. A common method to increase the total spin of such molecule is by engaging several metal centers in a strong ferromagnetic coupling. This strategy is applied in a recent study [1] of the tetranuclear transition metal compounds with formulas M₄(NP^tBu₃)₄ (M = Ni, Cu), and the oxidised forms, [M₄(NP^tBu₃)₄]⁺. These results suggest a strongly coupled, large-spin ground state in the two nickel compounds.

The two non-oxidised compounds, Ni₄(NP^tBu₃)₄ and Cu₄(NP^tBu₃)₄, are studied here with respect to the bonding interactions between the metal centers. X-ray diffraction experiments have been performed on crystals of both compounds at the synchrotron facility SPring-8 in Japan. Based on the data from the experiments, a multipole model of the charge density is achieved for both complexes. Theoretical structure factors are also calculated based on the experimental atomic positions of the complex containing nickel, and a theoretical multipole model is also developed.

A topological analysis is performed on all three datasets. The plots of the critical points in the charge density show no bond critical points between the metal centers as shown on Fig. 1. This indicates that no bonding is present between the metal centers, which is supported by the results from deformation density plots such as the one in Fig. 2. Plots of the critical points in the Laplacian for the nickel containing complex show that the charge density around the nickel atoms are affected by one another but not strongly, which is supported by calculations of the delocalization index.

Tautomeric systems such as keto-enol and enamine-imine incorporating both donor and acceptor in the same molecule, can be utilised to design single-component molecular ferroelectrics. This type of prototropic system reinforce intra or intermolecular hydrogen bonds synergistically with the augmentation of the π-system delocalization, defined as RAHB.¹ The ferroelectric systems developed utilizing the aforementioned phenomenon are defined as proton tautomerizarion type ferroelectrics and the corresponding mechanism is known as proton tautomerism mechanism (PTM).² However, this mechanism was never probed from electron density distributions. Here, we report the charge density analysis of 2-(4-(trifluoromethyl)phenyl)-1H-phenanthro [9,10-d] imidazole (1), the single-component molecular ferroelectric material (Figure 1).³ For elucidating the PTM in this molecular crystal, we have performed multipole model⁴ based experimental charge density analysis using high-resolution X-ray diffraction data. Thereby, we studied the deformation of electron densities (ED), the bond paths along with the bond critical points and the electrostatic potential (ESP) map along the N-H…N hydrogen bonds and performed topological analysis of the electron densities for understanding the underlying mechanism behind the proton transfer. The analysis also highlights the amphoteric characteristic of the enamine-imine unit (Figure 1). The different degrees of polarization of the electron densities of the N-H group and the N-atom of the adjacent molecule clearly supports the asymmetric N-H…N hydrogen bond characteristic in this displacive type ferroelectric. Such accurate analysis of multipole based electron densities further strengthen the understanding of the proton tautomerism effect in hydrogen bonded molecular ferroelectrics. To the best of our knowledge, this is the first report of charge density analysis on a molecular ferroelectric material. Our study points to the necessity of charge density analysis for improved understanding in this niche area of molecular ferroelectric research.

Figure 1: (left) P-E loop and remanent polarization and (right) PTM pathway highlighting in terms of ED and ESP in 1

[1] P. Gilli, V. Bertolasi, V. Ferretti, G. Gilli, J. Am. Chem. Soc. 1994, 116 (3), 909–915.

[2] (a) S. Horiuchi, K. Kobayashi, R. Kumai, S. Ishibashi, Nat. Commun. 2017, 8, 1–9. (b) S. Dutta, V. Vikas, A. Yadav, R. Boomishankar, A. Bala, V. Kumar, T. Chakraborty, S. Elizabeth and P. Munshi, Chem. Commun., 2019, 55, 9610–9613.

[3] S. Dutta, Vikas, T. Vijayakanth and P. Munshi, Ferroelectric and Negative Thermal Expansion Properties in a purely organic multifunctional material. 2021 (to be published).

[4] N. K. Hansen and P. Coppens, Acta Crystallogr. Sect. A 1978, 34 (6), 909–921.

In-situ crystallization is a powerful tool to obtain the structure of compounds that are liquid at room temperature [1]. The technique involves cooling the sample in a glass capillary below its liquid-solid phase transition temperature, initializing crystallization and using the crystalline powder so obtained as starting material for crystal growth. In favorable cases, a crystal suitable for X-ray analysis can be obtained at the liquid-solid phase boundary by translation of the capillary through the cold nitrogen gas stream (zone melting). If the quality of the in-situ grown crystal is sufficiently good, experimental charge density studies (resolution up to 0.5 Å) become possible. Nowadays there are many models used to describe the experimental diffraction intensities (IAM, multipole model, maximum entropy method etc.) [2]. With the appearance of quantum crystallographic methods (Hirshfeld Atom Refinement HAR and X-Ray Constrained Wavefunction analysis XCW) that combine the experimental results with quantum mechanical calculations, it is possible to get precise insights into the "true" nature of the wavefunction and the charge density of a molecule [3]. The charge density of a molecule can also be described as a superposition of theoretical calculated Invariom (Invariant atom) or directly by the theoretically calculated charge density [4]. But which of these methods is best suited for which scientific question or experimental resolution? Different charge density models, such as the multipole-model, HAR and the Invariom-approach, were investigated using experimental diffraction intensities of different in-situ crystallized liquids. The residual density in difference Fourier maps as used to evaluate the quality of the theoretical model (Fig. 1) [5]. It was found that multipole parameters derived from fitting to theoretically calculated charge density describe the experiment almost as well as unrestricted multipole refinement. In addition, using theoretical parameters the reflection to parameter ratio is improved.

Information on 3D elemental distribution is critically important in studies of catalysis, life sciences, materials research, toxicology, geology, and many other fields. To access this information X-rays are advantageous compared to electron microscopy and atom probe tomography techniques because of their short wavelengths, high penetrability and possibility to image samples non-destructively. X-ray fluorescence (XRF) tomography stands out among other synchrotron-based techniques for its high sensitivity to low elemental concentrations, simultaneous access to many elements in a single measurement, and for its compatibility with other simultaneous imaging modalities. Recent developments of nano-focussing optics for hard X-rays have advanced XRF tomography towards submicron resolution.
Although higher resolution can be easily achieved in 2D, practical limitations due to long scanning times and instrument instability and positioning accuracy have limited the 3D resolution to a few hundred nanometers [1, 2]. With expected improvements of brilliance at upgraded diffraction-limited storage rings, this limit will be pushed through the reduction of acquisition time. However, resolving the sample stability and positioning accuracy issue will immediately benefit existing XRF imaging stations (both for 2D and 3D case) and will have an even greater impact on the imaging quality of upgraded storage rings.
In this talk, we will present current developments in combining XRF tomography and ptychographic X-ray computed tomography (PXCT) at the cSAXS beamline of the Swiss Light Source. In this approach, we use flOMNI (flexible tOMography Nano Imaging, see Figure 1) [3], a unique instrument that allows accurate sample positioning with respect to the focusing optics by means of differential laser interferometry. The setup is now equipped with a fluorescence detector. Overcoming the stability issue gives us an opportunity to achieve resolution in XRF tomography on the level of the probe size with minimal data manipulation. While combining the electron density contrast from PXCT and the element specificity of fluorescence allows us to exercise various data analysis strategies that will be covered in this talk. We expect that this development will find interest from synchrotron users in fields ranging from life sciences to materials science.

[1] T. Victor et al., X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam, Sci. Rep. 8, 13415 (2018).
[2] G. Martínez-Criado et al., ID16B: a hard X-ray nanoprobe beamline at the ESRF for nano-analysis, J. Synchrotron Rad. 23, 344-352 (2016).
[3] M. Holler et al., X-ray ptychographic computed tomography at 16 nm isotropic 3D resolution, Sci. Rep. 4, 3857 (2014).

High-resolution imaging of 3D structure and elemental composition is critical for studies ranging from biology to materials science. ID16A is well up to the challenge with its established record in hard X-ray phase and fluorescence imaging. Two phase imaging modalities are routinely offered at the beamline: holography that explores longitudinal diversity and near-field ptychography that explores the transverse diversity. The recent upgrade of ESRF further improves the beamline performance through increased flux and coherence and reduced spectral bandwidth.

For phase imaging the improvements of the source can lead to higher resolution and better data quality (particularly in the near-field ptychography regime). It will also directly benefit X-ray fluorescence imaging where higher flux translates directly into increased elemental sensitivity and reduced acquisition times, allowing to survey more samples resulting in the higher statistical significance of the results.

In this poster, we will discuss the status of the beamline after the transition to the ESRF-EBS and its exciting implications for the user community. We will also discuss immediate plans in the beamline development where new collaborations with the users will be mutually beneficial.

Coherent imaging methods such as ptychography have a growing interest for multidimensional imaging applications due to the high spatial resolution at which quantitative information is obtained. In the application of X-ray imaging to the study of dynamic processes [1], the achievable temporal resolution is limited by detector performance. However, for ptychography this is also determined by the degree of redundancy in the diffraction data that is needed to reliably reconstruct real-space images. The success of ptychography lies in the incorporation of redundancy in the spatial dimensions from transverse translation diversity of the object, which allows for relaxation of constraints on the illumination and other experimental factors. Other schemes such as longitudinal translation (phase) diversity [2], and probe diversity [3], have been reported.

Using the time dimension redundancy for the introduction of a constraint has recently been proposed in coherent diffractive imaging (CDI) [4,5], exploiting “overlap” between successive images to achieve similar advantages to ptychography. However, these methods impose limits on the object or illumination and require a priori knowledge of the location of time-independent regions of the object, making them incompatible with scanning methods such as ptychography. We have developed an algorithm that removes these limitations and introduces redundancy without guidance and without requiring reference object regions by exploiting intentional or incidental temporal diversity in the diffraction data [6]. Our approach automatically defines a spatiotemporal constraint through automatically segmenting time-dependent and time-independent regions within the image field, dependent on the detected sample dynamics, and is able to suppress ambiguity and artefacts in the reconstructions.

Spatiotemporal redundancy in time-series coherent diffraction data provides a viable path toward studying nanoscale dynamics by X-ray imaging. We demonstrate this potential through CDI simulations of different dynamic phenomena under realistic conditions modelled on the XFM beamline at the Australian Synchrotron, the motion of nanoparticles, as well as the oscillatory behaviour of a 2-dimensional chemical reaction. An extension to dynamic ptychography that allows high quality reconstructions to be achieved with a relaxed spatial overlap constraint and faster scanning is then examined through simulation. Preliminary experimental investigation of crack propagation within thin metallic films, obtained using the EIGER2 detector at the XFM beamline, will be presented.

This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). This research was undertaken on the XFM beamline at the Australian Synchrotron, part of ANSTO.

[1] J. Lim et al., “Origin and hysteresis of lithium compositional spatiodynamics within battery primary particles,” Science, 353, 566 (2016).

[2] C. T. Putkunz, et al., “Phase-Diverse Coherent Diffractive Imaging: High Sensitivity with Low Dose,” Phys. Rev. Lett. 106, 013903 (2011).

[3] I. Peterson, et al., “Probe-diverse ptychography”, Ultramicroscopy 171, 77 (2016).

[4] Y. H. Lo, et al., “In situ coherent diffractive imaging,” Nat. Commun. 9, 1826 (2018)

[5] X. Tao, et al., “Spatially correlated coherent diffractive imaging method,” Appl. Opt. 57, 6527 (2018).

[6] G. N. Hinsley, et al., “Dynamic Coherent Diffractive Imaging Using Unsupervised Identification Of Spatiotemporal Constraints,” Opt. Express 28, 36862 (2020).

Coherent X-ray diffraction imaging (CXDI) is a powerful lensless imaging technique utilizing X-rays with a high degree of coherence [1]. Conceptually, CXDI can image isolated microscopic objects with high resolution and since its first demonstration in 1999 [2] it has laid the foundation for the development of other methodologies such as ptychography and Bragg CXDI [1]. With the new low-emittance storage ring [3] at ESRF combined with a state-of-the-art Eiger 4M detector and efficient iterative algorithms, the ID10 beamline is optimised for CXDI experiments. Three-dimensional imaging of crystalline and amorphous particles at ~14 nm resolution [4] has recently been demonstrated. In contrast to conventional characterisation methodologies such as electron microscopies, CXDI is ideally suited to study the surface morphology and interior of such microscopic particles without sectioning or ion-milling.

We used CXDI to image in 3D a series of CaCO₃ microparticles prepared under different crystallization and growth conditions, revealing that the microparticles systematically assume a wide range of morphologies as function of temperature [5]. In this presentation, we demonstrate 3D CXDI imaging of CaCO₃ particles 3-6 µm in diameter, with 16 nm voxel size. Wide-angle X-ray diffraction (WAXD) patterns [6] were recorded in combination with the CXDI datasets to identify the crystalline phase of the CaCO₃ microparticles and obtain information of characteristic crystal planes. Figure 1 shows the evolution of CaCO₃ particle morphology as a function of precipitation temperature: from the nested hexagonal morphology at T = 25˚ C to an appearance of spikes at T = 35˚, and finally transforming to an extended rod-shape morphology at T = 45˚ C. In addition, the internal structures of the particles and density variations within the particles can also be appreciated in 3D. Finally, we discuss the challenges arising due to radiation damage and how to resolve them, and also the future prospects of dynamic or serial CXDI experiments.

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[2] Miao, J., Charalambous, P., Kirz, J. & Sayre, D. (1999). Nature 400, 342.

[3] ESRF News. (2017). No. 77, December 2017.

[4] Cherkas, O., Beuvier, T., Breiby, D. W., Chuskhin, Y., Zontone, F. & Gibaud, A. (2017). Cryst. Growth Des. 17, 4183.

[5] Oral, M. Ç. & Ercan, B. (2018). Powder Technology. 339, 781.

[6] Chushkin, Y., Zontone, F., Cherkas, O. & Gibaud, A. (2019). J. Appl. Cryst. 52, 571.

For non-destructive imaging of three-dimensional (3D) materials and biological specimen, hard X-ray in-line holography is particularly suitable, since it offers a phase-sensitive imaging scheme which can cover large specimen in a full-field approach without the need for scanning. Unfortunately, the resolution of holographic imaging is limited by the source size of the cone-beam illumination, and does not reach values in the sub-20 nm regime which are routinely achieved by ptychography or CDI.

We have implemented holographic X-ray imaging based on cone-beam illumination, beyond the resolution limit given by the cone-beam numerical aperture. In this new single-shot approach [1], image information encoded in the far-field diffraction and in the holographic self-interference is treated in a common reconstruction scheme, without the usual empty beam correction step of in-line holography. An illumination profile tailored by waveguide optics and exactly known by prior ptychographic probe retrieval is shown to be sufficient for solving the phase problem. We demonstrate the improved experimental capability by reconstruction of a test pattern with a field of view of 5×5µm² and a resolution of 11 nm, using a waveguide exit source size of about 30 nm (FWHM).

Figure 1 shows the divergent illumination (a) from the waveguide exit (b) to the sample plane (c). To quantify the resolution we have analyzed the reconstruction of a pattern with 50 nm (half-period) lines and spaces (d,e). The reconstruction by the presented method shows higher resolution and image quality compared to conventional single-shot reconstruction by the contrast-transfer-function approach after empty-beam division. The resolution of the new reconstruction approach was determined by Fourier ring correlation (FRC) indicating a resolution (half-period) of Δ = 11.2 nm (f).

The approach paves the way towards high resolution and dose-efficient X-ray tomography, well suited for the current upgrades of synchrotron radiation sources to diffraction limited storage rings.

The three-dimensional X-ray diffraction (3dxrd) technique provides a useful tool to investigate polycrystalline materials, grain-by-grain, in a non-destructive way. The approach of the scanning 3dxrd microscopy is to probe the sample by moving a pencil beam horizontally across it (y direction) with a resolution dependent on the beam size. For each step, the sample is rotated of 180° (or 360°, ω angle) in order to collect the diffraction spots of all the grains in the sample [1].

We used a combined approach of scanning 3dxrd and Phase Contrast Tomography (PCT) to investigate the hydration of a widespread hydraulic binder material, namely gypsum plaster. This material forms when the bassanite (calcium sulfate hemihydrate) reacts with water. In-situ 3dxrd measurements allowed to understand the crystallographic lattice, orientation and position of each grain in the sample during the hydration reaction (Figure 1 a,b).

The PCT reconstructions, instead, allowed the visualization of the shape of the crystals in the sample over time and a quantification of density and porosity (Figure 1 c,d).

Monitoring the evolution of the hydration reaction of gypsum plaster with both these techniques appears to be a promising tool to gain insights about the kinetics of the hydration reaction, the crystallization and growth of the hydrated phase and the shape of the final gypsum crystals that build the interlocked and porous gypsum plaster hardened mass.

X-Ray Photon Correlation Spectroscopy (XPCS) is a flexible tool to quantify dynamics on the nanometer to micrometer scale in bulk samples and was used in the recent years in grazing incidence (GI) geometry for application to thin film samples, such as quantifying thin film growth [1]. Measurements in GI geometry introduce distortions of the detected signal. These distortions are due to refraction and reflection and known from the Distorted Wave Born Approximation (DWBA), which leads to superpositions of signal within detector areas [2]. Zhang et al. [3] showed that these superpositions also influence GI-XPCS measurements and can alter observation quantities like decorrelation times and stretching exponents. We present an approach to quantify the influence these refraction and reflection effects due to the DWBA have on decorrelation analysis by conducting grazing incidence transmission (GT) XPCS and Gi-XPCS simultaneously for a thin film sample of Methyl Ammonium Leadiodide, showing non-equilibrium dynamics. A combination of the GI- and GT XPCS results with calculations of Fresnel reflectivities and transmissivities within the simplified DWBA allows to determine the origin of scattering contributions for GT and GI regions. Considering calculations of the non-linear effect of refraction in GISAXS and GTSAXS, comparable regions to XPCS experiments in transmission are identified and differences for phenomena like altered decorrelation times and decay stretching are elucidated. This allows the use of this technique to analyze dynamics in thin films for certain experimental conditions. [4]
[1] Headrick, R. L., Ulbrandt, J. G., Myint, P., Wan, J., Li, Y., Fluerasu, A., Zhang, Y., Wiegart, L. & Ludwig, J. K. F. (2019). Nature Communications. 10, 1–9.
[2] Lu, X., Yager, K. G., Johnston, D., Black, C. T. & Ocko, B. M. (2013). Journal of Applied Crystallography. 46, 165–172.
[3] Zhang, Z., Ding, J., Ocko, B. M., Fluerasu, A., Wiegart, L., Zhang, Y., Kobrak, M., Tian, Y., Zhang, H., Lhermitte, J., et al. (2019). Physical Review E. 100, 1–8.
[4] Greve, C. R., Kuhn, M., Eller, F., Buchhorn, M. A., Kumar, D., Hexemer, A., Freychet, G., Wiegart, L. & Herzig, E. M., manuscript in preparation.

Functional properties such as dielectric constant [1] and hydrogen storage [2] in fine crystalline materials often exhibit particle size effects. Understanding the phenomena with size effects and utilizing their functions require observing the structure in conjunction with shape, size, and heterogeneity information.

We report the development and improvement of an apparatus for Bragg coherent x-ray diffraction imaging (Bragg-CDI) [3] at BL22XU in SPring-8. The achieved observable particle size was 40–500 nm and Pd (~40 nm) and ferroelectric barium titanate (BaTiO₃, BTO, 200~500 nm) fine crystals were investigated.

This study aims to achieve two primary goals. (1) The first is to reduce background noise due to x-ray scattering by air. To this end, we newly prepared a vacuum chamber for samples, enabling us to obtain high-contrast x-ray diffraction pattern for a shorter time. (2) The second is to optimize a real-space constraint; our modified phase-retrieval algorithm can use appropriate real-space constraints with shrinking [4] support to refine the phase distribution.

We succeeded in expanding the observable particle-size range from 100–300 [3] to 40–500 nm [5] for the Bragg-CDI at BL22XU in SPring-8. The reconstructed three-dimensional image showed the outer shape, size, and internal phase (strain) for a single particle. A single 500-nm BTO particle showed a straight and sharp antiphase-boundary shape, whereas smaller BTO particles showed different phase boundary shapes. The present Bragg-CDI thus allows the observation of the outer shape, size, and inner phase distribution for a single particle with a size of tens to hundreds of nanometres, which may lead to a simple understanding of mesoscale ferroelectricity.

This work was partly supported by JSPS Grant-in-Aid for Scientific Research (Grant Nos. JP19H02618, JP18H03850, JP18H05518, JP19H05819, JP19H05625) and The Murata Science Foundation.

Full author list: N. Oshime, K. Ohwada, K. Sugawara, T. Abe, R. Yamauchi, T. Ueno, A. Machida, T. Watanuki, S. Ueno, I. Fujii, S. Wada, R. Sato, T. Teranishi, M. Yamauchi, K. Ishii, H. Toyokawa, K. Momma and Y. Kuroiwa.

[1] T. Hoshina, J. Ceram. Soc. Jpn. 121, 156 (2013). [2] M. Yamauchi, R. Ikeda, H. Kitagawa, and M. Takata, J Phys C, 112, 3294 (2008). [3] K. Ohwada, K. Sugawara, T. Abe, T. Ueno, A. Machida, T. Watanuki, S. Ueno, I. Fujii, S. Wada, and Y. Kuroiwa, Jpn. J. Appl. Phys. 58, SLLA05 (2019). [4] S. Marchesini, H. He, H. Chapman, S. Hau-Riege, A. Noy, M. Howells, U. Weierstall, and J. Spence, Phys. Rev. B 68, 140101 (2003). [5] N. Oshime, K. Ohwada, K. Sugawara, T. Abe, R. Yamauchi, T. Ueno, A. Machida, T. Watanuki, S. Ueno, I. Fujii, S. Wada, R. Sato, T. Teranishi, M. Yamauchi, K. Ishii, H. Toyokawa, K. Momma and Y. Kuroiwa, Jpn. J. Appl. Phys. (in press). https://doi.org/10.35848/1347-4065/ac148b

Interpretation and analysis of XFEL data can depend critically on a fundamental understanding of the characteristics of the XFEL pulses. To exploit the unique repetition rate of the EuXFEL requires understanding of both the inter- and intra-train fluctuations in pulse fluence, spatial energy distribution, coherence and wavefront, and beam pointing, which are frequently implicated in the loss of information in XFEL single particle imaging (SPI) and other classes of coherent diffraction experiment. Failure to account for fluctuations of the electron bunch phase-space and/or trajectory within a pulse train can result in deviations of the recorded wavefront, intensity statistics and intensity integrals from theoretical behaviour.
Preliminary X-ray optical data collected at the SPB-SFX instrument of the European XFEL demonstrates a sensitivity of inter- and intra-train variations in beam pointing to different beam delivery parameters (pulses-per train). We present this data alongside a model of the SASE1 photon beam. A partially coherent wave optical simulation of the beam propagated from the undulator exit to the SPB-SFX instrument hutch is compared to experimental data collected in the same plane. Also discussed will be the design of wavefront measurement methods that can be made for comparison with theory. Moving forward, we outline a novel method for investigating the relationships between the statistical behaviour of the XFEL source (including inter- and intra-train jitters) and the optical (wavefront) properties observed at the instrument to extend these observations.

Coherent diffraction imaging (CDI) allows for sub-wavelength spatial resolution by reconstructing an image from recorded diffraction patterns using a phase-retrieval algorithm [1, 2]. CDI is e particularly advantageous in the extreme ultraviolet (EUV) and X-ray ranges, where optics manufacturing is difficult and expensive [2]. Ptychography, the scanning version of CDI, has several benefits, such as large field of view imaging and robustness. The sample (object) is scanned by moving a spatially confined illumination (probe) while ensuring overlap in the illuminated regions [3]. The complex object function is typically retrieved by using an iterative algorithm that relies on two constraints [4]. First, the real space (or overlap) constraint assumes that the exit wave leaving the sample is formed by the probe function multiplied with the object function, i.e. the thin object approximation [3]. Second, the Fourier constraint enforces the estimated diffraction pattern intensity to match the measured diffraction data.

To optimize the reconstruction procedure, additional constraints have been suggested, based on a priori knowledge of the object and the measurement system. For example, Guizar-Sicairos et al. [5] have proposed a statistical optimal reconstruction procedure that finds the solution to the ptychographic problem by a least-squares approximation of the maximum likelihood function. Alternatively, an approach by Katkovnik et al. [6] that uses a sparse approximation of the probe and object function additionally to a maximum likelihood technique, to improve the reconstruction quality compared to a non-optimized algorithm. Recently, Ansuinelli et al. [7] have directly used the sample’s layout information to build an optimal reconstruction algorithm for imaging a photolithography mask, by penalizing the deviation of the reconstructed mask image to a full mask model.

We present here a phase-retrieval algorithm similar to Chang et al. [8] and Enfedaque et al. [9] that solves the blind ptychography problem (retrieving the probe and object) using total variation regularization (TV) as an additional constraint on the object function. TV promotes a sparse object gradient and is therefore preferential for (quasi) binary structures, removing noise and image artefacts [10]. We will discuss the total variation based algorithm for EUV photolithography mask inspection and show the impact of the algorithm for reconstruction of simulated and experimental data.

[1] Chapman, H. N. & Nugent, K. A. (2010). Nature Photonics, 12, pp. 833 – 830.

[2] Gardner, D., Tanksalvala, M., Shanblatt, E.R., et al. (2017). Nature Photonics, 11, pp. 259 – 263.

[3] Rodenburg, J. & Faulkner, H. (2004). Appl. Phys. Lett., 85, pp. 4795 – 4797.

[4] Thibault, P., Dierolf, M., Bunk, O., et al. (2009). Ultramicroscopy, 109, pp. 338 – 343.

[5] Guizar-Sicairos, M. & Fienup, J. (2008). Optics Express, 16, pp. 7264 – 7282.

[6] Katkovnik, V. & Astola, J. (2012). J. Opt. Soc. Am. A, 30, pp. 367 – 379.

[7] Ansuinelli, P., Coene, W. M. J. & Urbach, H. P. (2020). Applied Optics, 59, pp. 5937 – 5947.

[8] Chang, H., Enfedaque, P. & Marchesini, S. (2019). 2019 IEEE International Conference on Image Processing, pp. 2931 – 2935.

[9] Enfedaque, P., Chang, H., Krishnan, H., et al. (2018). Computational Science – ICCS 2018, pp. 540 – 553.

[10] Rudin, L. I., Osher, S. & Fatemi, E. (1992). Physica D, 60, pp. 259 – 268.