RNA-Puzzles is a collective endeavour dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. Systematic protocols for comparing models and crystal structures are described and analyzed. In RNA-Puzzles, we discuss a) the capabilities and limitations of current methods of 3D RNA structure based on sequences; b) the progress in RNA structure prediction; c) the possible bottlenecks that hold back the field; d) the comparison between the automated web server and human experts; e) the prediction rules, such as coaxial stacking; f) the prediction of structural details and ligand binding; g) the development of novel prediction methods; and h) the potential improvements to be made.

Till now, 28 RNAs with crystal structures have been predicted, while many of them have achieved high accuracy in comparison with the crystal structures. We have summarized part of our results in three papers and two community-wide meetings. With the results in RNA-Puzzles, we illustrate that the current bottlenecks in the field may lie in the prediction of non-Watson-Crick interactions and the reconstruction of the global topology. Correct coaxial stacking and tertiary contacts are key for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution.

We now further extend the prediction to RNA sequences in the Rfam families. We have predicted structures for 20 RNA families, while some of the predictions have been confirmed by crystal or cryo-EM structures, indicating the possibility to use predicted models for functional inference. The predicted models also helped in 'Molecular Replacement' for crystal structures.

For the model evaluation, we present RNA-Puzzles toolkit, a computational resource including (i) decoy sets generated by different RNA 3D structure prediction methods (raw, for-evaluation and standardized datasets), (ii) 3D structure normalization, analysis, manipulation, visualization tools (RNA_format, RNA_normalizer, rna-tools) and (iii) 3D structure comparison metric tools (RNAQUA, MCQ4Structures).

With the increasing number of RNA structures being solved as well as the high-throughput biochemical experiments, RNA 3D structure prediction is becoming routine and accurate. Experimental data-aided structure modelling may effectively help in understanding the noncoding RNA function, especially the viral RNAs.

The experimental models archived in the Protein Data Bank provide a rich source of structural information on proteins and nucleic acids. Complex architectures of RNA molecules as well as non-canonical DNA structures prove that the sugar-phosphate backbone is not a scaffold-like structure more or less passively accommodating to and enabling base pairing and stacking motifs formed by the four nitrogenous bases. In the past, RNA structures attracted more attention [1-5] as their 3D folds are formed by visibly rich ensemble of the backbone geometries. The self-recognition of DNA duplexes posed seemingly fewer challenges to analysis of their structural details. However, a detailed look showed structurally well defined conformers [6, 7] that proved useful in discriminating different modes of binding of DNA to transcription factors and the nucleosome core particle in histones [8]. The analysis has shown that differences in the local DNA structure relate to specificity of the binding. In the year 2020, the conformational spaces of DNA and RNA, which were traditionally analyzed separately, were described by one unified set of dinucleotide conformers, which are called NtC, and by a related structural alphabet CANA, Conformational Alphabet of Nucleic Acids [9]. I will briefly describe the principle of fully automated and robust assignment of the NtC classes and CANA symbols and overview related tools that help to annotate, validate, refine experimental structures, and build computer models of NA molecules. All these tools are feely available at the web service dnatco.datmos.org [10].
[1] Duarte et al. NAR 31:4755 (2003). [2] Murray et al. PNAS USA 100:13904 (2003). [3] Hershkovitz et al. NAR 31:6249 (2003). [4] Schneider et al. NAR 32:1666 (2004). [5] Richardson et al. RNA 14:465 (2008). [6] Svozil et al. NAR 36:3690 (2008). [7] Schneider et al. Acta Cryst D74:52 (2018). [8] Schneider et al. Genes 8:278 (2017). [9] Černý et al. NAR 48:6367 (2020). [10] Černý et al. Acta Cryst D76:805 (2020).

Recent developments in the field of evolutionary covariance and machine learning have enabled the precise prediction of residue-residue contacts and increasingly accurate inter-residue distance predictions. Access to accurate covariance information has played a pivotal role in the recent advances observed in the field of protein bioinformatics, particularly the improvement of prediction of protein folds by ab initio protein modelling. As this work seeks to showcase, this data is of equal value in the field of X-ray crystallography, with several practical applications in MR, model validation and map interpretation.

The most prevalent technique for the solution of the phase problem in macromolecular crystallography is molecular replacement (MR). In most cases, the availability and detection of a suitable search model, typically a solved structure homologous to the target of interest, is the key limitation of conventional MR. In those cases where no such structure is available, unconventional MR approaches are used. Recent results suggest that even in those cases where no homologous structures are found for a given target, it may still be possible to find suitable search models among unrelated structures, in the form of regions that share high, albeit local, structural similarity with the target. The challenge then becomes the accurate identification of such search models among the vast number of available solved structures. Here we present SWAMP, a novel pipeline for the solution of structures of transmembrane proteins, which exploits the latest advances in residue contact predictions for the detection of fragments later to be used as search models. SWAMP includes a library of ensembles built by clustering commonly observed packings of transmembrane helical pairs in close contact, mined from the available databases. Residue contact predictions are used in the process of search model selection: the contact maximum overlap between the target’s predicted contacts and the observed contacts of each member of the library is used to estimate the likelihood of the helical pair being a successful search model. Preliminary results show that SWAMP is capable of detecting valid search models originating from unrelated solved structures solely exploiting this contact information. This enables the solution of new and challenging structures without the use of experimental phasing techniques, and opens a whole new avenue of research in which predicted contact information is used to extend the reach of unconventional MR.

The final outcome of X-ray crystallographic experiments is the determination of the structure of interest, which requires building a model that satisfies the experimental observations. However, experimental limitations can lead to the presence of unavoidable uncertainties during model building resulting in regions that require validation and potentially further refinement. Many metrics are available for model validation, but are mostly limited to the consideration of the physico-chemical aspects of the model or its match to the map. We present new metrics based on the availability of accurate inter-residue distance predictions, which are then compared with the distances observed in the emerging model. Early results suggest that these metrics are capable of detection of register and other errors, even in challenging cases where conventional metrics may struggle.

Residue contact and inter-residue distance predictions are usually represented respectively as two-dimensional binary matrices called contact maps and distograms. These typically omit contacts between sequential near neighbours resulting in a blank space on and near the diagonal axis of the matrix. A multitude of properties can be predicted by other sequence-based methods and researchers often need to consider diverse sources of information in order to form a complete and integrated picture for the inference of structural features that can facilitate the structure solution. Here we present ConPlot, a web-based application which uses the typically empty space near the contact map or distogram diagonal to display multiple coloured tracks representing other sequence-based predictions. These predictions can be uploaded in various popular file formats. This web application is currently available online at www.conplot.org, along with documentation and examples.

I will present some recent developments of our Pepsi package for integrative modeling of macromolecules guided by small-angle scattering profiles. These include very fast tools for the all-atom computations of X-ray and neutron small-angle scattering profiles, called Pepsi-SAXS and Pepsi-SANS, respectively [1,2]. These tools implement algorithms specifically designed to handle two notable properties of large macromolecules and their complexes, such as for instance viral capsids, namely their high flexibility and high degree of symmetry. Flexibility of macromolecules is not spontaneous but linked with their structure and function. Computationally, it can be often approximated with just a few collective coordinates, which can be computed e.g. using the Normal Mode Analysis (NMA). NMA determines low-frequency motions at a very low computational cost and these are particularly interesting to the structural biology community because they give insight into protein function and dynamics. On our side, we have proposed a computationally efficient nonlinear NMA method that can be applied to largest complexes from the Protein Data Bank (PDB), and which also very well preserves local stereochemistry [3-5].

Flexibility of macromolecules is often linked with their structure and function. Computationally, it can be approximated with just a few collective coordinates computed using the Normal Mode Analysis (NMA). NMA determines low-frequency motions at a very low computational cost. This technique is particularly interesting for the structural biology community as it allows extrapolating biologically relevant motions starting from high-resolution structures. Recently, we have shown that it can be extended to model local deformations and to better preserve the stereochemistry of the protein. We have developed a computationally efficient nonlinear NMA method that can be applied to the largest complexes from the Protein Data Bank (PDB) [3-5].

Large symmetrical protein structures have seemingly evolved in many organisms because they carry specific morphological and functional advantages compared to small individual protein molecules. Recently we have proposed a novel free-docking method for protein complexes with arbitrary point-group symmetry [6]. It assembles complexes with cyclic symmetry, dihedral symmetry, and also those of high order (tetrahedral, octahedral, and icosahedral). We also proposed an efficient analytical solution to the inverse problem, that is the identification of symmetry group with the corresponding axes and their continuous symmetry measures in a protein assembly [7-8].

With Pepsi-SAXS and Pepsi-SANS, one can leverage the above-mentioned developments, by optimizing structures along low-frequency « normal modes », performing automatic and adaptive coarse-graining of molecular models, rescoring free-docking predictions, including those of symmetric assemblies, and also optimizing structural transitions. Structural models produced by Pepsi-SAXS/SANS were ranked top in the recent data-assisted protein structure prediction sub-challenge in CASP13 [9].

[1] Grudinin, S. et al. (2017). Acta Cryst. D, D73, 449 – 464. For more information https://team.inria.fr/nano-d/software/pepsi-saxs/
[2] https://team.inria.fr/nano-d/software/pepsi-sans/
[3] Hoffmann, A. & Grudinin, S. (2017). J. Chem. Theory Comput. 13, 2123 – 2134. For more information https://team.inria.fr/nano-d/software/nolb-normal-modes/
[4] Grudinin, S., Laine, E., & Hoffmann, A. (2020). Predicting protein functional motions: an old recipe with a new twist. Biophysical journal, 118(10), 2513-2525.
[5] Laine, E., & Grudinin, S. (2021). HOPMA: Boosting protein functional dynamics with colored contact maps. The Journal of Physical Chemistry B, 125(10), 2577-2588.
[6] Ritchie, D. W. & Grudinin, S (2016). J. Appl. Cryst., 49, 1-10.
[7] Pages, G., Kinzina, E, & Grudinin, S (2018). J. Struct.Biology, 203 (2), 142-148.
[8] Pages, G. & Grudinin, S (2018). J. Struct.Biology, 203 (3), 185-194.
[9] Hura, G. L., ... & Tsutakawa, S. E. (2019). Small angle X‐ray scattering‐assisted protein structure prediction in CASP13 and emergence of solution structure differences. Proteins: Structure, Function, and Bioinformatics, 87(12), 1298-1314.

Displacement parameters (B-factors) play a crucial role in macromolecular structure determination, yet are rarely used for biological interpretation. This is somewhat egregious, since they account for the local flexibility of individual protein states/conformations. We have developed a new approach^[1] for dividing the disorder information in a macromolecular model into a hierarchical series of components on different length-scales, which reveals the components of the atomic disorder that result from molecular disorder, domain disorder, or local atomic disorder. This makes both molecular and atomic disorder intuitively understandable in terms of likely domain motions and internal atomic motions. We demonstrate this new approach by studying the flexibility of the catalytic site in crystal structures of the SARS-CoV-2 main protease. Additionally, we apply the method to structures determined by cryo-EM, where we can investigate and visualize the flexibility in both the extended and non-extended receptor-binding domains of the SARS-COV-2 spike glycoprotein, and in the iron-reductase STEAP4, which hint at a mechanism for electron transfer.

Ribonucleic acid (RNA) molecules are master regulators of cells. They are involved in many molecular processes: they transmit genetic information, sense cellular signals and communicate responses, and even catalyze chemical reactions. RNA function and in particular its ability to interact with other molecules such as proteins, is encoded in the sequence. Understanding how RNAs and RNA-protein complexes carry out their biological roles requires detailed knowledge of the RNA structure.

Due to limitations in experimental structure determination, complete high-resolution structures are available for a tiny fraction of all the known RNA molecules crucial for numerous fundamental cellular processes. <1% of RCSB entries represent RNA structures, and only around 3% of RNA families available in the Rfam database have at least one experimentally determined structure. This relative paucity of information compared to what is available for proteins also makes computational RNA 3D structure prediction much less successful. Currently, purely computational RNA 3D structure prediction is limited to sequences shorter than 100 nt.

I will present strategies for computational modeling of RNA and RNA-protein complex structures that utilize SimRNA, a suite of methods developed in my laboratory, which use coarse-grained representations of molecules, rely on the Monte Carlo method for sampling the conformational space, and employ statistical potentials to approximate the energy and identify conformations that correspond to biologically relevant structures. In particular, I will discuss the use of computational approaches for RNA structure determination based on low-resolution experimental data, including low-resolution crystallographic electron density maps and cryo-EM maps.

References

1 Ponce-Salvatierra, A. et al. Biosci. Rep. 39, BSR20180430 (2019)
2 Boniecki, M. J. et al. Nucleic Acids Res. 44, e63 (2016)

The inflammasome signaling pathways are activated by infections and sterile stimulation, which lead to the maturation of inflammatory caspases that promote the secretion of inflammatory cytokines such as IL-1b and IL-18. The recognition and cleavage of the gasdermin family members by caspases trigger the activation of their pore-forming activities that lead to pyroptotic cell death. A prominent example is the targeting of gasdermin D (GSDMD) by inflammatory caspases-1/4/5/11 as an essential step in initiating pyroptosis following inflammasome activation. Previous work has identified cleavage site signatures in caspase substrates such as GSDMD and inflammatory cytokines, but it is unclear if these are the sole determinants for caspase engagement. Here we describe structural studies of a complex between caspase-1 (CASP1) and the full-length GSDMD, which reveals that the cleavage site-containing linker in GSDMD adopts a long loop structure that engages the CASP1 active site. In addition, an exosite is observed between the caspase-1 L2 and L2’ loops and a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. The exosites endows a novel function for the GSDMD C-terminal domain as a caspase-recruitment module, in addition to its role in autoinhibition. The dual site recognition may allow stringent substrate selectivity while facilitating efficient cleavage and pyroptosis upon inflammasome activation. Such mode of tertiary structure recognition may be applicable to other physiological substrates of caspases.

Plants and animals detect pathogen infection via intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Structure-function studies of these complexes are hampered by low levels in native tissue, our inability to express them recombinantly, and their instability in solution. We overcame sample limitation problems and solved a 3.8 Å resolution cryo-EM structure of the activated ROQ1, an NLR native to N. benthamiana with a Toll-like interleukin-1 receptor (TIR) domain, bound to the Xanthomonas effector XopQ. ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effectors to trigger cell death and confer disease resistance. The Toll/interleukin-1 receptor (TIR) domains of plant NLRs can hydrolyze nicotinamide adenine dinucleotide in its oxidized form (NAD+), which is required for NLR-mediated immune signaling. The Cryo-EM structures of the RPP1 and Roq1 resistosomes show that formation of two asymmetric dimers of TIR domains is critical for the NADase activity. However, the structural mechanism underlying TIR-catalyzed NAD+ cleavage remains unknown. Here, we report a crystal structure of RPP1-TIR in complex with NAD+. The TIR domain forms a tetramer in an asymmetric unit, which is nearly identical with that seen in the RPP1 resistosome. The NAD+ is bound to the catalytic center between the asymmetric head-to-tail TIR homodimers, with the adenosine group contacting one TIR monomer (TIRb) and the phosphate groups and the nicotinamide ribose contacting the other TIR (TIRa). The nicotinamide-ribose bond of NAD+ has been cleaved, and the free nicotinamide stacks against the adenosine group. The carboxylate oxygen of the catalytic Glu158 interacts with the C-2 and C-3 hydroxyl groups of the nicotinamide ribose, and the interactions are highly conserved in the cADPR-bound ADP-ribosyl cyclase CD38. Our study reveals NAD+ recognition mechanism of a plant TIR domain and provides insight into NAD+ hydrolysis catalyzed by the TIR protein.

Most T cells found in jawed vertebrates express functional heterodimeric receptors (TCRs) on their surface formed by either α and β or γ and δ chains. Each chain possesses two domains, an amino-terminal variable domain (V) and a constant domain (C) on the carboxy-terminus (V-C pattern). In most cases, the ability of T cells to recognize diverse antigens relies on the surface (or paratope) located within Vα – Vβ or Vγ – Vδ segments. Recent genomic studies of non-eutherian mammals identified clusters of genes that resemble the classical TCR loci but surprisingly contain an additional variable segment. The functional product common for marsupials and monotremes called ‘μ chain’ was predicted to contain two variable (Vμ and Vμj) and one constant (Cμ) domains. Single cells analysis of blood and spleen from Monodelphis domestica showed that some of the splenic T cells co-express the μ and γ chains suggesting that both polypeptides could form a novel type of T cell receptor, the γμTCR. Using obtained sequences, we generated and structurally characterized two different γμTCRs. Here, we present the novel and unusual architecture of a third lineage of T cell receptor found in marsupials and monotremes [1].

[1] Morrissey K.A., Wegrecki M., Praveena T., Hansen V.L., Bu L., Sivaraman K.K., Darko S., Douek D.C., Rossjohn J., Miller R.D., Le Nours J. The molecular assembly of the marsupial T cell receptor defines a third T cell lineage. SCIENCE, 371, 1383-1388, 2021.

Human surfactant protein D is a collectin and member of the C-type lectin superfamily of proteins that forms an essential part of the mammalian innate immune system. The collectins have been recognised to not only bind to invading pathogens, allowing for recognition by immune cells, but also play an important role in activating and regulating the response of both the innate and acquired immune systems. Crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of human SP-D (hSP-D) complexed with the inner core oligosaccharides from gram negative bacterial human pathogens Haemophilus influenzae, Salmonella enterica sv Minnesota rough strains [1-2] and Escherichia coli provide unique multiple insights into the recognition and binding of bacterial lipopolysaccharide (LPS) by hSP-D. LPS binding is achieved through calcium dependent recognition of the proximal inner core heptose dihydroxyethyl side chain coupled with specific interactions with the binding site flanking residues Arg343 and Asp325 and evidence for an extended binding site for LPS inner cores. Where this preferred mode of binding is precluded by the crystal lattice, oligosaccharide is bound through a terminal core glucose.

The structures thus reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved heptose (Hep) with the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes including terminal or non-terminal sugars, when the preferred inner core Hep is not available for binding. Alongside binding studies of both whole bacteria and LPS the structures also demonstrate that carbohydrate extensions to the core LPS oligosaccharide, previously thought to be targets for collectins, are important in shielding the more vulnerable target sites in the LPS core. Recent structures of hSP-D bound with small synthetic LPS core component ligands which include phosphorylation of specific carbohydrate residues further demonstrate not only the ability to adopt alternative modes of recognition but also that LPS phosphorylation can provide an additional mechanism by which pathogens can efficiently evade a first-line mucosal innate immune defence.

[1] Clark, H. W., Mackay, R. M., Deadman, M. E., Hood, D. W., Madsen, J., Moxon, E. R., Townsend, J. P., Reid, K. B. M., Ahmed, A., Shaw, A. J., Greenhough, T. J. & Shrive, A. K. (2016). Infection & Immunity 84, 1585.

[2] Littlejohn, J. R., da Silva, R. F., Neale, W. A., Smallcombe, C. C., Clark, H. W., Mackay, R. M. A., Watson, A. S., Madsen, J., Hood, D. W., Burns, I., Greenhough, T. J. & Shrive, A. K. (2018). PLOS One 13, e0199175.

Metabolite based T cell immunity is emerging as a major player in antimicrobial immunity, autoimmunity and cancer. Here, small molecule metabolites were identified to be captured and presented by the major histocompatibility complex (MHC) class-I related molecule MR1 to T cells, namely Mucosal Associated Invariant T cells (MAIT) and diverse MR1-restricted T cells. Both MR1 and MAIT T cell receptors (TCR) are evolutionarily conserved in many mammals, suggesting important roles in host immunity. Namely, during infection with riboflavin-producing microorganisms, MR1 trapped riboflavin-based metabolites and presented on the surface of the antigen-presenting cells encountering the MAIT TCR leading to the activation of the MAIT cells. How modifications to these small molecule-metabolites affect presentation by MR1 and MAIT cell activation remains unclear.
To dissect the molecular basis underpinning MR1 antigen capture and MAIT recognition, we chemically synthesized and characterized a large panel of these naturally occurring metabolites, termed “altered metabolite ligands” (AMLs), and investigated functionally and structurally their impact on the MR1-MAIT axis. Through the generation and detailed analysis of 13 high-resolution MAIT TCR-MR1-AML crystal structures, along with biochemical and functional assays, we show that the propensity of MR1-upregulation on the cell surface was related to the nature of MR1-AML interactions. MR1-AML adaptability and a dynamic compensatory interplay at the TCR-AML-MR1 interface impacted the affinity of the TCR-MR1-AML interaction, which ultimately underscored the ability of the AMLs to activate MAIT cells. Here, I will provide a generalized framework for metabolite recognition and modulation of MAIT cells. Further, I will discuss the design, use and implications of “AMLs” for selective and specific tailoring of T cell immune responses.

1. Awad, W. ^#, Ler, G.M.^# et al. (2020). The molecular basis underpinning the potency and specificity of MAIT cell antigens. Nature Immunology 21, 400-411.
2. Salio, M.^#, Awad, W.^# et al. (2020). Ligand-dependent downregulation of MR1 cell surface expression. PNAS, 117(19), 10465–10475.
3. Awad W, et al. (2020). Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1. J. Biol. Chem. 295(42), 14445–14457.

The first stable binary icosahedral quasicrystals (iQCs) were found in Cd-Yb and Cd-Ca alloy systems [1, 2], which was followed by finding of nine isostructural iQCs in Cd-R (R = Y, Gd-Tm) [3] and Zn-Sc systems [4]. The structural type of these iQC is called Tsai-type, and it has been extended to ternary or quaternary alloys by atomic substitutions.

Higher-dimensional structure analysis of the Cd-Yb iQC by single-crystal X-ray diffraction revealed that the atomic structure consists of two main building blocks, rhombic triacontahedron (RT) and acute rhombohedron (AR) units [5]. In ternary iQCs, the structure analysis becomes more difficult because occupational disorder has to be taken into account in the 6D structure model. Furthermore, recent studies have shown that some sites are preferentialy occupied by the substituting elements [6,7], which indicates that the higher-dimensional structure model must be optimized to ternary iQCs. To build such model, knowledge of atomic structures in ternary quasicrystal approximants (APs) is quite important.

In the first part of my talk, I will present the superstructure and basic structure of ternary Yb-Cd-Mg 1/1APs with the compositions Yb_12.9Cd_78.4Mg_8.8 and Yb_13.3Cd_64.2Mg_22.5 [8]. The former was determined to have a face-centred packing structure comprising two distinguishable RT units (space group Fd3, a = 31.377(1) Å), while the latter was found to have a body-centred packing structure made of identical RT units (space group Im3, a = 15.7596(4) Å). The distinction between the two types of RT units in the superstructure is based on the positional disorder of the first tetrahedron shell and the relative Cd/Mg occupancy at sites (48h) in the fourth icosidodecahedron shell.

In the second part, I will introduce a Python package (PyQCstrc) for building the higher-dimensional models of iQCs [9] and present a modification of six-dimensional structural model for the primitive Tsai-type iQCs so as to incorporate the selective Cd/Mg occupation found in the Cd-Mg-Yb 1/1 APs [7].

[1] A.P. Tsai, J.Q. Guo, E. Abe, H. Takakura, and T.J. Sato, (2000), Nature, 408, 537–538.
[2] Guo, J. Q., Abe, E., Tsai, A. P. Phys. Rev. B. (2000), 62, R14605−R14608.
[3] Goldman, A. I., Kong, T., Kreyssig, A., Jesche, A., Ramazanoglu, M., Dennis, K. W., Bud’ko, S. L., Canfield, P. C., (2013), Nat. Mater, 12, 714−718.
[4] Canfield, P. C., Caudle, M. L., Ho, C. S., Kreyssig, A., Nandi, S., Kim, M. G., ... & Goldman, A. I. (2010), Phys. Rev. B, 81(2), 020201.
[5] Takakura, H., Pay Gómez, C., Yamamoto, A., de Boissieu, M., and Tsai, A.P., (2007), Nat. Mater. 6, 58–63.
[6] Pay Gómez, C. & Tsai, A. P. (2013). Comptes Rendus Physique, 15(1), 1–10.
[7] Yamada, T., Takakura, H., de Boissieu, M. and Tsai, A.-P., (2017), Acta Cryst. B73, 1125-1141.
[8] Yamada, T, (2021), Phil. Mag., 101(3), 257-275.
[9] Yamada, T, J. Appl. Cryst., in press.

Keywords: Quasicrystal, Approximant

This work was supported by JSPS KAKENHI grants (numbers JP18K13987, JP19H05818).

It has been known for some time now that normally a crucial element in QC formation, at least in soft matter, is the presence of two prominent wave numbers in the linear response behaviour to periodic modulations of the particle density distribution. This is equivalent to having two prominent peaks in the static structure factor or in the dispersion relation. In the first half of the talk, we demonstrate how the crucial pair of prominent wave numbers are connected to the length and energy scales present in the pair potentials. Whilst the ratio between the two length scales is important, we show here that for thermodynamically stable soft matter quasicrystals, the ratio of these wave numbers should be close to certain special values. We identify features in the particle pair interaction potentials which can suppress or encourage density modes with wave numbers associated with one of the regular crystalline orderings that compete with quasicrystals, enabling either the enhancement or suppression of quasicrystals. In the second half of the talk we look how to compute phase diagrams for a given interaction potential in an efficient manner. In order to do this, we focus on the representation of the density distribution in soft matter systems. The form of the average (probability) density distribution in solids is often represented as a sum of Gaussian peaks (or similar functions) centred on lattice sites or via a Fourier sum. Here, we argue that representing instead the logarithm of the density distribution via a Fourier sum is better. The advantage of this representation is that it excels both deep in the crystalline region of the phase diagram and also close to melting. Additionally, we show how a strongly nonlinear theory (SNLT) enables efficient computation of the phase diagram for a threedimensional quasicrystal-forming system using an accurate nonlocal density functional theory.

The atomic structure of the decagonal Al-Cu-Rh quasicrystal with a space group is refined based on five X-ray diffraction datasets, collected at 293 K, 1013 K, 1083 K, 1153 K and 1223 K with the use of a synchrotron radiation [1]. The real-space structure solution with the tiling-and-decoration approach based on the moment series expansion [2] is executed.

All the crystallographic – factors are ranging from 5.9% to 6.4% for the datasets of common 1460 symmetry-inequivalent peaks. What is the most intriguing is the correlation (Pearson correlation equal to 0.85) between lattice parameters (edge-length of rhombus and the interatomic layer distance) and the maximum of the residual electron density. The identical temperature dependence presented in figure 1 for the parameters implies the phase transformation. The residual density is agglomerated in the origin of the 4D unit cell what implies the phase transformation is related to the General Penrose Tiling (GPT). Additionally, we can observe a local minimum around the 1083-1153 K of the moments values being directly related to phasons. This is the temperature the structure is the most stable around. The existence of the local minimum in all the plots proves the phason disorder is related to the structure stability what was previously questioned due to insufficient quality of the refinement [1].

We modified the moment series approach to accommodate the existence of the 5^th atomic surface arising for the random-tiling model of the decagonal quasicrystal (figure 2). After the structural refinement with the updated model we obtained much better results in terms of the R-factors. Even more, up to the uncertainty estimated with Hesse matrix, we could prove the 5^th atomic surface existence is not only the artefact of the electron density calculation but the crucial feature of the structure in the 1083-1153 K. The calculations prove the random-tiling hypothesis of the structural stability is true for the decagonal quasicrystals and the structure is stabilized by phasons.

Beyond Golay-Rudin-Shapiro S. I. Ben-Abrahamshelomo.benabraham@gmail.com

I briefly recapitulate the necessary background about the original pseudorandom Golay-Rudin-Shapiro sequence (GRS) and its known generalizations [1-5]. The standard method to make the one-sided GRS based on a two-letter alphabet A₂ = {a, b} two-sided is by constructing a proto-GRS structure based on a four-letter alphabet A₄ = {a, b, c, d} and then reduce it to A₂. In order to generalize to higher dimensions one proceeds analogically. Here I extend GRS to eight symbols (alias letters, digits or colors). I also refine the terminology introducing the designation dD GRSn for a GRS structure based on n symbols and supported by Z^d.

The most natural support for 3D GRS8, that is a structure is based on A₈ = {0, 1, 2, 3, 4, 5, 6, 7} is Z³. The respective substitution is

(1)

The bottom matrix refers to 2D GRS8. The bottom line, in turn, refers to 1D GRS4, while the alphabet A₈ splits into two disjoint A₄'s. Thus 1D necessitates special treatment. As in the case of GRS4, the substitution must be applied twice.

Fig.1 shows an isometric projection of the hull of the second generation of 3D GRS8.

Figure 1. Aspect of hull of 3D GRS8 generation 2.The Fourier spectrum of all GRS structures is absolutely continuous [7, 8].

[1] Golay, M. J. E. (1949) J. Opt. Soc. Amer. 39 437-444.

[2] Rudin, W. (1959) Proc. Amer. Math. Soc. 10 855-859.

[3] Shapiro, H. S. (1951) Extremal problems for polynomials and power series, Master's thesis (MIT, Cambridge MA).

[4] Queffélec, M. (1995) Substitution dynamical systems – spectral analysis, LNM 1294, 2nd. ed. (Springer Verlag, Berlin).

[5] Ben-Abraham, S. I. and David, A. (2020) J. Phys.: Conf. Ser. (in press).

[6] Allouche J.-P. and Shallit J. (2003) Automatic Sequences: Theory, Applications, Generalizations, (Cambridge University Press.

[7] Baake, M. and Grimm, U. (2013) Aperiodic Order. Volume 1: A Mathematical Invitation, (Cambridge University Press).

[8] Barbé, A. and von Haeseler, F. (2003) J. Phys. A: Math. Gen. 38 2599-2622.

Keywords: Golay-Rudin-Shapiro structures

Developing realistic three-dimensional growth models for quasicrystals is a fundamental requirement. Uchida found a general principle for building crystal structures (the Uchida stacking motif) in complex alloys such as the μ-Al₄Mn phase [1]. Here, we investigated the Uchida stacking motif using molecular dynamics (MD) simulations to search for clues to the origins of the atomic arrangements in quasicrystals. We used the LAMMPS code for the MD simulations. Our MD simulation results well reproduce the Uchida stacking motif seen in the μ-Al₄Mn phase. The simulations also reveal the formation of a deformed icosahedron. Our results provide new insights into the growth mechanism and origin of complex alloys and quasicrystals.

[1] Uchida, M. & Matsui. Y. (2000). Acta Cryst. B56, 654.

The atomic-resolution holography (ARH) technique [1,2] offers the possibility to experimentally determine the local atomic-scale structure of quasicrystals. This method can selectively investigate specific elements and their 3-dimensional local atomic environment in a range of up to around 2 nm, without the need of a priori information on the structure. Therefore, it can provide a novel perspective for the visualization of the structure of aperiodic systems.

Recently, we have described the results of the ARH reconstruction for the Penrose lattice, which can be regarded as a reference system for decagonal quasicrystals. The resulting pattern of atomic images can be interpreted a projection of the average structure.[3] Using this framework, we can now describe how the experimental results for decagonal Al-Co-Ni quasicrystals compare with the projection of the average structure from a computational model.[4]

An example is shown in the Figure below, with exemplary data of an experimental hologram of an Al-Co-Ni quasicrystal (a). The intense lines in the hologram are the so-called X-ray standing wave lines, which indicate the 10-fold symmetry of the system. The reconstruction of the environment around the Co atoms from the holograms is illustrated in (b), and is compared with the corresponding projection from the computational model (c). Shown here is the quasi-periodic plane that includes the emitter atom at the origin. The atomic images at the vertices of the dashed polygons can be identified with transition metal atoms, while Al atoms are mainly distributed along the polygon edges.

We will also demonstrate the differences of the quasiperiodic structure versus a crystalline approximant and illustrate the ARH results for icosahedral structures.

Small Angle Neutron Scattering is a low-resolution technique enabling to probe the solution structure of individual biomacromolecules possibly in complex with its partners. In particular, concerning membrane proteins, the membrane-like environment can be made invisible in order to see only the protein. Here, we combined SANS with X-ray crystallography, cryoEM, H/D exchange coupled with mass spectrometry and limited proteolysis to reveal the flexibility and ligand-induced conformational changes of the multidrug ABC transporter BmrA.

Limited proteolysis revealed an important flexibility of BmrA WT in most steps of its catalytic cycle. Cryo-EM provided high-resolution of the closed conformation by analysis of an artificially monodisperse sample, and X-ray crystallography data enabled to build homology models of other conformations, which constituted the starting point of SANS analysis. H/D X-MS pinpointed the flexible part along the transporter sequence and SANS revealed the extent of this flexibility.

Together, these techniques enable us to describe the ABC transporter cycle in term of successive conformational equilibria, a much more realistic and accurate vision of this biological process [1].

Figure 1. A: Main steps of the enzymatic cycle of ABC transporters (from [2]); B: Structural definition of these steps in solution by sequential conformational equilibria [1].

[1] Javed et al. in preparation

[2] Wannes Dermauw, Thomas Van Leeuwen, The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance, Insect Biochemistry and Molecular Biology, Volume 45, 2014

Molecular dynamics (MD) is crucially important for protein functions. MD simulation is a powerful computational tool for investigating molecular dynamics of proteins in atom detail. However, due to the time-scale limitation of MD simulation, conformational samplings in MD simulation are occasionally insufficient. Thus, to validate simulation structures, the comparison of the simulation structures with experimental results is useful.

Small-angle x-ray scattering (SAXS) experiments is a powerful method to measure protein structures in solution. Although the resolution of SAXS is limited to low because of the orientational and conformational averaging, the information of protein conformations in solution can be obtained. Therefore, the comparison of simulation results with SAXS data serves to obtain the protein solution structures consistent with experiments.

We have developed a hybrid method of MD simulations and SAXS (MD-SAXS) [1­–3]. The first example of MD-SAXS applications was EcoO109I, a type II restriction endonuclease [1]. The enzyme was revealed to be substantially flexible, and the intrinsic flexibility was found to be closely related to the structural changes upon DNA binding.

Ion effects on SAXS data were investigated using MD-SAXS [2]. At a series of ion concentrations from 0 to 1 M, the MD-SAXS analysis for lysozyme was performed. The SAXS excess intensities were strongly dependent on ion concentrations. Based on the MD-SAXS, we developed a fast method to handle ion effects.

MD-SAXS was also applied to the drug target protein [4]. Vitamin D receptor (VDR) is a member of the nuclear receptor family, and functions as the control of the expression of genes through Vitamin D binding. The VDR ligand binding domain (LBD) is expected to undergo conformational changes upon agonist or antagonist binding. However, the crystal structures of VDR-LBD share a similar structure even with bound agonist or antagonist. The crystal structure of VDR-LBD in the ligand-free state has not been determined. The SAXS experiments suggest that both the ligand-free and antagonist-bound structures in solution are different from the crystal structure. Thus, the MD-SAXS analysis was performed to elucidate the solution structures of VDR-LBD in both the states. In the ligand-free and antagonist-bound state, the obtained solution structures were in good agreement with their SAXS data. Their structural features were consistent with the function of VDR.

Sampling capability of all-atom MD simulations is occasionally insufficient for very flexible and large molecules. To overcome the limitation, we developed a hybrid method of a coarse-grained MD simulations and SAXS (CG-MD-SAXS) [5]. Even in the coarse-grained models (e.g., Cα only), SAXS data were accurately reproduced from the structure models. CG-MD-SAXS was applied to the three types of nucleosomes (canonical, CENP-A, and H2A.B nucleosomes), and revealed the substantial difference in the dynamics of DNA around histones.

[1] Oroguchi, T., Hashimoto, H., Shimizu, T., Sato, M., Ikeguchi, M. (2009) Biophys. J. 96, 2808.

[2] Oroguchi, T., Ikeguchi, M. (2011) J. Chem. Phys. 134, 025102.

[3] Oroguchi, T., Ikeguchi, M. (2012) Chem. Phys. Lett. 541, 117.

[4] Anami, Y., Shimizu, N., Ekimoto, T., Egawa, D., Itoh, T., Ikeguchi, M., Yamamoto, K. (2016) J. Med. Chem. 59, 7888.

[5] Ekimoto, T., Kokabu, Y., Oroguchi, T., Ikeguchi, M. (2019) Biophys. Physicobiol. 16, 377.

: The RNA transcription complex (RTC) from the virus, SARS-CoV-2, is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities the RTC mechanism must also conform to a large number of imperatives including RNA over DNA base recognition, base pairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interface with error checking machinery and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8 and Nsp12 (also known as RNA dependent RNA polymerase (RdRp)). We have solved high resolution crystal structures of the Nsp7/8 complex providing insight into the interaction between the proteins. We have used small angle X-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher order complexes and with and without RNA. Using size exclusion chromatography and multi-angle light scattering coupled SAXS (SEC-MALS-SAXS) we defined which combination of components form transient or stable complexes. We used contrast matching neutron scattering to mask specific complex forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8 and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS and other biophysical techniques we provide greater insight into the RTC assembly, mechanism and potential avenues for disruption of the complex and its functions.

Correct formation of disulfide bonds is critical to the folding of a wide variety of proteins. Bacterial virulence factors are one class of proteins containing disulfide bonds, thus, an approach to disarm virulent bacterial might involve shutting down the machinery involved in the formation of disulfide bonds. The suppressor of copper sensitivity (Scs) proteins form part of the disulfide bond forming machinery in bacteria, and it is hoped that determining the structure of molecules such as this may lead to the development of new classes of antibiotics. There are four Scs proteins (ScsA, B, C and D) present in numerous Gram-negative bacteria, and few have been structurally characterised. In this work we show that the ScsC protein from Proteus mirabilis is trimeric and flexible, where the high level of flexibility is afforded by a glutamine rich motif. We also show that the protein interacts with ScsB and that this interaction rigidifies the ScsC protein.

Despite their importance in function, the conformational states and changes of proteins are often poorly understood mainly because of the lack of an efficient tool. MurD, a 47-kDa three-domain protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during its catalytic cycle are not well understood. The previous crystallographic studies have identified two major conformational states of MurD, open and closed conformations, in which the domain 3 has distinct orientations with respect to the other two domains. The conformational difference between the two crystal structures suggested that MurD can undergo drastic conformational changes in solution. However, the details about the conformational states and changes of MurD in solution coupled with the binding with the ligands or the inhibitors remained to be elucidated.

In our study, we exploited multiple biophysical methods including nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation to demonstrate evaluation of the conformational states and distribution of MurD. We exploited paramagnetic lanthanide ions that can be attached to the specific position(s) on the protein by the use of the lanthanide tags [1]. In NMR, the effects of the paramagnetic lanthanide ions are observed as pseudo-contact shifts (PCSs) that can provide long-range (< ~40 Å) distance and angular information of each of the observed nuclei in the protein [1]. The lanthanide ion was fixed on the domain 2 of MurD and PCSs were observed from the resonances derived from the domain 3. Analysis of PCSs achieved estimation of conformational states of MurD in solution and detection of the conformational changes of MurD induced by its ligands and inhibitors [2]. The paramagnetic lanthanide ions, especially gadolinium ions, can be exploited by EPR and double electron–electron resonance (DEER) measurement that provides inter-gadolinium distance and population (distance distribution) [1]. The distance distributions obtained from DEER measurement were consistent with the information derived from PCS-NMR, SAXS, and MD simulation.

Our study highlights several biophysical methods to investigate the overall conformational states of a multi-domain protein. The integrated use of these methods can be an efficient strategy to evaluate the conformational states and distribution of proteins in solution.

[1] Saio, T., Ishimori, K. (2020) Biochim. Biophys. Acta. Gen. Subj. 1864.

[2] Saio, T., Ogura, K., Kumeta, H., Kobashigawa, Y., Shimizu, K., Yokochi, M., Kodama, K., Yamaguchi, H., Tsujishita, H., Inagaki, F. (2015) Sci. Rep. 5, 16685.

Small-angle X-ray scattering (SAXS) experiments provide an estimation of biological macromolecule geometry on the level of domain structure. The reliability of structural inference drawn from SAXS data is dependent on the accurate measurement as well as the proper post-processing procedure. The methods improving raw data quality and gaining more information are widely explored. Among those innovations, size-exclusion chromatography small-angle X-ray solution scattering (SEC-SAXS) has become a standard method for modern bio-SAXS synchrotron light sources (Ryan et al. 2018; Brennich et al. 2016; Blanchet et al. 2015). However, the principle of data post-processing for SEC-SAXS remains rather unclear. This includes background correction and averaging of the raw data. Several statistical tools have been developed to assess solution SAXS data quality (Rambo and Tainer 2013; Franke et al. 2015). These are mostly useful for “rejecting” significantly different data points or data frames, based on the assumption that the rest of the data are close to the “truth”. But this can lead to a situation where mediocre data, for example data contaminated with radiation damage, are not correctable or simply cannot be evaluated before any interpretation is done.

To alleviate this problem, an objective metric, correction-state score (CSS) is proposed. CSS can be used to both verify the data quality and identify the optimal data correction procedure for post-processing of SEC-SAXS data. CSS can be represented as a numerical likelihood with a scale of 0 to 1. Using this objective score it is possible to quantitatively assess the “goodness” or appropriateness of a background correction for SEC-SAXS data. Under the guidance of CSS, the metadata recorded during a SEC-SAXS experiment can be used to maximise the fidelity of the post-processing as well as reduce the ambiguity in further data interpretation.

References
Blanchet, C.E., Spilotros, A., Schwemmer, F., et al. 2015. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). Journal of Applied Crystallography 48(Pt 2), pp. 431–443.
Brennich, M.E., Kieffer, J., Bonamis, G., et al. 2016. Online data analysis at the ESRF bioSAXS beamline, BM29. Journal of Applied Crystallography 49(1), pp. 203–212.
Franke, D., Jeffries, C.M. and Svergun, D.I. 2015. Correlation Map, a goodness-of-fit test for one-dimensional X-ray scattering spectra. Nature Methods 12(5), pp. 419–422.
Rambo, R.P. and Tainer, J.A. 2013. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496(7446), pp. 477–481.
Ryan, T.M., Trewhella, J., Murphy, J.M., et al. 2018. An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis. Journal of Applied Crystallography 51(1), pp. 97–111.

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.

My laboratory studies how molecules on the surface of prokaryotic cells mediate cellular interaction with the environment, enabling cellular motility, initiating cellular adhesion to surfaces, and facilitating biofilm formation. For our work, we leverage our expertise in electron cryotomography (cryo-ET) in situ imaging, together with ongoing method development in subtomogram averaging approaches for structure determination of macromolecules in their native context. We combine cryo-EM with FIB milling of specimens and cryo-light microscopy to study molecules on prokaryotic cells.

In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk. FATZ proteins interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub.
Here we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose a molecular interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organise interaction partners and stabilise α-actinin-2 preferential orientation in the Z-disk.
Finally, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the intriguing question whether FATZ proteins can create an interaction hub for Z-disk proteins through membrane-less compartmentalization during myofibrillogenesis.

Bacterial RNA polymerase (RNAP) is an essential multisubunit enzyme performing transcription. Regulation of this process is secured through the stage-dependent interactions of RNAP with different factors (mostly proteins). Here we report the structure-function analysis of the functional complexes between RNAP and a unique helicase-like factor HelD [1] which is present in many Gram-positive bacteria (e.g. Bacillus subtilis and Mycobacterium smegmatis) [2, 3]. HelD forms tightly bound complexes with RNAP. It simultaneously penetrates into RNAP primary and secondary channels which are responsible for nucleic acids binding and substrate delivery. HelD can also interact with the RNAP active site. Structurally, these interactions are incompatible with the binding of DNA to the RNAP core and thus with the elongation stage of transcription. This is in accordance to our functional data showing that HelD is capable of clearing RNAP of nucleic acids and that HelD can dismantle RNAP-DNA complexes. HelD itself is composed of several domains, showing structural changes in solution [2] as well as in complexes with RNAP (three different structural states obtained from the cryo-EM analysis) [3]. Although we were able to link the observed dynamic behaviour with the DNA-clearing role of HelD, the recycling of HelD-bound RNAP and subsequent restart of transcription remains to be explained.

HelD as well as its complexes with RNAP resisted our attempts to crystallize them for many years. In order to get to the structural details we took the advantage of recent developments in the field of single-particle cryo-EM and were able to obtain ~3Å resolution structures. The structure of HelD itself was completely unknown with no homologue in the PDB. We combined X-ray crystallography (structure of one domain) and cryo-EM, together with bioinformatics and homologous modelling and successfully built de novo a complete atomic model of the HelD protein. For the analysis of condition-dependent dynamic behaviour we used small-angle X-ray scattering [2]. Results from our structural studies were supplemented with biochemical and biophysical assays (enzymology, analysis of interactions and stability) and by computational analyses [3].

[1] Wiedermannová, J., Sudzinová, P., Kovaľ, T., Rabatinová, A., Šanderova, H., Ramaniuk, O., Rittich, Š. & Dohnálek, J. (2014). Nucleic Acids Res. 42, 5151-5163.

[2] Kovaľ, T., Sudzinová, P., Perháčová, T., Trundová, M., Skálová, T., Fejfarová, K., Šanderová, H., Krásný, L., Dušková, J. & Dohnálek, J. (2019). FEBS Lett. 593, 996-1005.

[3] Kouba, T., Koval', T., Sudzinová, P., Pospíšil, J., Brezovská, B., Hnilicová, J., Šanderová, H., Janoušková, M., Šiková, M., Halada, P., Sýkora, M., Barvík, I., Nováček, J., Trundová, M., Dušková, J., Skálová, T., Chon, U., Murakami, K.S., Dohnálek, J. & Krásný, L. (2020). Nat Commun.11, 6419.

This work was supported by MEYS (LM2015043 and CZ.1.05/1.1.00/02.0109), CSF (20-12109S and 20-07473S), NIH (grant R35 GM131860), AS CR (86652036), ERDF (CZ.02.1.01/0.0/0.0/16_013/0001776 and CZ.02.1.01/0.0/0.0/15_003/0000447), EMBL (EI3POD) and Marie Skłodowska-Curie grant (664726).

Anomalous small-angle X-ray scattering (ASAXS) utilizes the changes of the scattering patterns emerging due to the variation of the scattering amplitude of a particular atom type upon changing the X-ray wavelength in the vicinity of the absorption edge of the atom. ASAXS on biological macromolecules is challenging due to the weak anomalous scattering effect. Biological macromolecules are also prone to radiation damage and often only available in small quantities, which further complicates the ASAXS measurements. First biological ASAXS experiments were done in the early 80-s at the European Molecular Biology Laboratory (EMBL) beamlines of the synchrotron DESY in Hamburg on metallo-proteins [1] but overall, ASAXS was not widely used in biological studies.

Recent progress in synchrotron instrumentation and dramatic increase of the brilliance of modern synchrotron sources revitalized the interest to biological ASAXS. The biological SAXS beamline P12 operated by EMBL at PETRA III storage ring (DESY, Hamburg) is dedicated to study macromolecular solutions [2] and allows for ASAXS on macromolecular solutions. The beamline was adapted to accommodate the needs for ASAXS by implementing careful data collection and reduction procedures and we report the recent developments at P12 allowing to conduct ASAXS on different macromolecular systems. Examples of utilizing ASAXS on various systems including, in particular, surfactants, nanoparticles, polymers and metal-loaded proteins are presented. The beamline control, data acquisition and data reduction pipeline developed for ASAXS on P12 are now available as standard tools for the biological SAXS community.

[1] H. B. Stuhrmann, Q. Rev. Biophys. 14, 433 (1981).

[2] C. E. Blanchet et al., J. Appl. Crystallogr. 48, 431 (2015).

Bacterial flagella are self-assembling nanomachines that enable cell motility by connecting a rotary motor to a long filament. Members of the Spirochaetes phylum, which includes important pathogens, swim using body undulations powered by the rotation of supercoiled flagella that remain confined within the periplasmic space and wrap around the cell body [1]. This behaviour diverges from that of other bacteria, where flagella function as extracellular propellers [2]. Spirochetal filaments are correspondingly distinct in composition and organization, but their molecular structure has remained elusive, obscuring the underlying mechanism for locomotion.

Here we show that, unlike all other known bacterial flagella, a highly asymmetric sheath layer coats the flagellar filament of the Leptospira spirochete and enforces filament supercoiling [3]. We solved 3D structures of wild-type and mutant flagellar filaments from L. biflexa, by integrating customized cryo-electron tomographic averaging methods with crystallographic analyses of sheath components FcpA and FcpB (Fig. 1). A central core made by FlaB (flagellin-like) protein units, delimits a central ~2nm pore.

Surrounding the core, FcpA and FcpB proteins colocalize exclusively on the outer, convex side of the filament, as an asymmetric sheath. Distinct sheath components, likely corresponding to FlaA isoforms, instead localize to the concave, inner side of the appendage. Sheath proteins were shown to produce filament supercoiling, an essential feature for flagellar-driven motility [1]. Such radial asymmetry represents a new paradigm of bacterial flagellar architecture, promoting filament supercoiling and ultimately enabling periplasmic flagella to exert their function in Leptospira translational motility. Given the large conservation of flagellar proteins across the Phylum, this asymmetric filament paradigm might prove valid for other spirochetes.

Candida albicans is the most common commensal fungus colonizing humans, and normally it does not impact the human health. However under certain conditions, it can rapidly outgrow bacterial flora causing mucocutaneous or systemic (and potentially fatal) infections. In most (mild) cases the treatment with topical and oral medications works well, however the resistant strains of C. albicans appear at the alarming pace, requiring the prompt development of new medications targeting this pathogen. One of the most promising routes to fight pathogens is to interfere with their protein synthesis machinery, therefore the structural information on ribosomes from pathogenic organisms is essential.

In this research we used an integrative structural biology approach based on the combination of single-particle cryo-Electron microscopy and macromolecular X-ray crystallography to resolve the structure of C. albicans ribosome.

We obtained 2.4 Å resolution structure of the 80S ribosome from C. albicans with the bound antibiotic and 4.2 Å resolution structure of the vacant C. albicans ribosome by single particle cryo-EM and X-ray crystallography. The comparison with other available eukaryotic ribosomes revealed unique features of C. albicans. These results can be used as a structural basis to decipher the mechanisms of antifungal resistance in C. albicans and to design novel inhibitors.

Single-Molecule Magnets (SMMs) are coordination compounds that exhibit slow relaxation of magnetization of molecular origin. In the case when SMMs contain only one paramagnetic metal center we distinguish the group of so-called Single-Ion Magnets (SMMs) [1]. In SIMs, the occurrence of slow relaxation of magnetization is closely related to the existence of non-negligible magnetic anisotropy on the magnetic center of the molecule. Since the magnetic anisotropy is strongly influenced by the topology and strength of the applied ligand field it could be expected that significant prolongation of coordination bonds or occurrence of non-covalent interactions involving the metal center may have a fundamental impact on resulting magnetic properties.

In 2016, we reported on static and dynamic magnetic properties of compound [Co(dpt)(NCS)₂], (dpt = 1,7-diamino-4-azaheptane). Two non-covalent interactions between the Co(II) atoms and π electrons of NCS^- ligands from the neighboring complex molecule (d(C···NC_centroid) = 3.55 Å) caused a mediation of ferromagnetic exchange interaction within the centrosymmetric dimer and also the dynamic magnetic properties were affected markedly [2]. This inspired us to investigate in greater detail the magnetic properties of Co(II) compounds having some of their metal-ligand bonds at distances longer than typical coordination bonds.

Semicordination bond can be considered as a non-covalent analogue of the coordination bond, which occurs when a weak attractive non-covalent interaction between an electrophilic region (associated with a metal center) and a nucleophilic region (associated with a nonmetal atom in another or in the same molecular entity) is formed [3,4]. In typical semicoordination bonds, the distances between the metal atoms and electron-donating groups are significantly longer than the sum of their covalent radii but shorter than the sum of van der Waals radii, the interactions are dominantly of electrostatic character and topology of electron density between the particular atoms exhibits bond path and critical point [4].

In line with the above-mentioned considerations, we chose to investigate three different series of mononuclear Co(II) compounds: (a) [Co(2NH₂-R^₁-py)₂(R^₂COO)₂], where R^₁ = H, 3/4/5-CH₃, R^₂ = CH₃, C₆H₅, t-Bu, the carboxylate ligand form Co-O bonds with lengths of 2.0 – 3.1 Å, (b) [Co(bq)(NO₃)₂(ROH)], where bq is 2,2'-biquinoline and ROH are various alcohol ligands, one of the nitrate ligands forms the Co-O bond with lengths of 2.5 – 3.3 Å, (c) [Co(R-pymep)₂], where H-R-pymep are various derivatives of 2-{(E)-[(pyridin-2-yl)imino]methyl}phenol, two Co-N bonds with lengths between 2.5 and 2.7 Å. We studied these compounds by a combination of experimental (X-ray diffraction, magnetometry, HF-EPR) and theoretical (DFT, CASSCF, Electronic localization function, non-covalent interaction index, and QTAIM) methods. In this talk, we report on the character of semicoordination in these compounds and the relationship between the structure and observed magnetic properties.

[1] Craig, G.A., Murrie, M. (2015). Chem. Soc. Rev. 44, 2135-2147.

[2] Nemec, I. et al. (2016). Dalton Trans. 31, 12479–12482.

[3] Ananyev, I.V. et al. (2020). Acta Cryst. B. 76, 436-449

[4] Efimenko, Z. M. et al. (2020). Inorg. Chem. 59, 2316–2327.

C-H and Si-H bond activation by metal-hydrogen bonding (agostic interactions) plays a central role in catalytic processes [1]. These processes are directly dependent on metal-hydrogen bond energies. The versatility of the coordination modes of the heavy metals allows wide structure and topology variations of the complexes. Therefore, it is of major importance to accurately describe these chemical bonds.

One important drawback is the difficulty of deriving accurate and precise hydrogen atom positions by any kind of experiment. Neutron-diffraction experiments would be the only reliable source of such information, but there is a lack of available accurate X-H bond distances with X being a transition metal from neutron diffraction. Therefore, it would be desirable to determine both the elongation of the C-H and Si-H bonds in agostic interactions and the metal-hydrogen bonding parameters from standard X-ray diffraction experiments. In this context, the capabilities of Hirshfeld Atom Refinement [2] to obtain precise and accurate C-H/Si-H…X bond parameters (with X=transition metal) are tested.

Experimental and theoretical charge densities of agostic interactions involving transition metal compounds have been determined and analyzed in the past [3]. Here, we use a combination of HAR with subsequent X-ray constrained wavefunction fitting [4] and purely theoretical calculations on the accurate HAR and neutron geometries to analyze the related chemical bonding beyond a charge-density analysis. We use three different test systems: Figure 1a shows Si-H…Cu/Ag interactions enforced through the ligands used by proximity constraints. We discuss whether there are signatures of agostic interactions in these systems with closed-shell (d¹⁰) coinage metal atoms. [5] Figure 1b shows a system where the proximity enforcing ligands have caused an oxidative addition reaction so that the hydrogen atom is now more closely bonded to the transition metal in a Rh-H…Si interaction. We analyze again to which extent (inverse) agostic interactions are present in this system. [6] Our findings will be referenced against classical C-H…Ti agostic interactions found in titanium amides (Figure 1c).[7]

Figure 1. Compounds (a) 1·MCl (M = Cu, Ag). Metal hydrides (b) RhH, (c) Titanium amide compounds.

[1] Bäckvall, J. E. (2002). J. Organomet. Chem. 652(1-2), 105-111.

[2] Jayatilaka, D., Dittrich, B. (2008). Acta Cryst. A, 64, 383-393.

[3] (a) Scherer, W., Wolstenholme, D. J., Herz, V., Eickerling, G., Bruck, A., Benndorf, P., Roesky, P. W. (2010). Angew. Chem., Int. Ed., 49, 2242-2246. (b) Hauf, C.; Barquera-Lozada, J. E.; Meixner, P.; Eickerling, G.; Altmannshofer, S.; Stalke, D.; Zell, T.; Schmidt, D.; Radius, U.; Scherer, W. (2013). Z. Anorg. Allg. Chem., 639, 1996-2004.

[4] (a) Jayatilaka, D. (1998). Phys. Rev. Lett. 80, 798-801. (b) Jayatilaka, D., Grimwood, D. J. (2001). Acta Cryst. A, 57, 76-86.

[5] Hupf, E., Malaspina, L. A., Holsten, S., Kleemiss, F., Edwards, A. J., Price, J. R., Kozich, V., Heyne, K., Mebs, S., Grabowsky, S., Beckmann, J. (2019). Inorg. Chem., 58 (24), 16372-16378.

[6] Holsten, S., Malaspina, L.A., Mebs, S., Hupf, E., Grabowsky, S., Beckmann, J. (2021) Organometalics. Under revision.

[7] Adler, C., Bekurdts, A., Haase, D., Saak, W., Schmidtmann, M., & Beckhaus, R. (2014). Eur. J. Inorg. Chem., 8, 1289-1302.

Single-ion magnets (SIMs) are a class of metal-organic coordination complexes with the intriguing ability to sustain a magnetic moment after the removal of a magnetizing field [1]. This ability originates in orbital angular momentum of unpaired electrons, introducing magnetic anisotropy that increases the magnetic relaxation time of the SIM magnetic moment. Magnetic anisotropy is therefore a key property in the search for new and improved SIMs, and it is imperative to be able to measure magnetic anisotropy experimentally.

In 2002, it was shown that polarized neutron diffraction from single crystals (PND) could be used to obtain information on so called ionic site-susceptibilities [2], which are tensor quantities that show the response of the magnetic moment of the ion to an external magnetic field. Site susceptibilities give direct access to the magnetic anisotropy of a compound, and we have earlier used this technique to measure the magnetic anisotropy of both lanthanide and transition metal SIMs in single crystals [3, 4]. Importantly, the technique was recently extended for application to powder samples [5].

Utilizing this exciting development, we have performed polarized neutron powder diffraction (pPND) on the SIM CoCl₂(tmtu)₂, tmtu=tetramethylthiourea (1). The compound shows zero-field splitting and slow relaxation of its magnetic moment [6], both requirements for a SIM. With pPND we obtain the orientation of the magnetic anisotropy with respect to the molecular structure (Fig. 1), and follow its dependence on magnetic field strength, directly from a powder sample. Comparison with PND measured on a single crystal of the Br-analogue CoBr₂(tmtu)₂ (2) shows that the powder and single crystal techniques give comparable results.

In this contribution, I will discuss the site susceptibility model, the application of the model to both single crystal and powder data and the magneto-structural correlations that we obtain from these measurements. The extension of the technique to powders, and the dramatic reduction in data acquisition times that it entails, means that compounds can be studied, for which the growth of suitably sized crystals for single crystal neutron diffraction is unattainable. This opens the possibility for magnetic anisotropy studies on a much wider range of molecular magnetic compounds under a larger range of experimental conditions.

Metal-Organic Frameworks (MOFs) are a class of crystalline organic-inorganic hybrid materials derived from metal nodes and organic linkers, that exhibit features like high surface area, well-defined pore, tunable structures and their properties [1]. Use of redox-active metal nodes or organic linkers, stable radical based ligands can introduce a special feature like conductivity, electrocatalyst, electrochromic behavior in MOFs apart from their conventional uses such as gas storage, gas separation, etc. This idea is impeded mainly due to the insulating nature of organic linkers and the instability of the framework to the redox process. This hindered the study of electroactive MOFs until the last decade. Recent advancement in this field has directed a surge of interest in understanding their mechanism of charge transfer. MOFs are a unique platform to investigate the charge transfer mechanism where the corresponding metal ions or organic linkers are well defined in a highly crystalline rigid system. Charge transfer is directed by either the through-space or through-bond approach [2]. The through-bond mixed-valance charge transfer has been well explored whereas, through-space intervalency in MOF is rare [3].

We have synthesized a new Cobalt (II) based metal-organic framework using redox-active organic linker, N,N′-di(4-pyridyl)thiazolo-[5,4-d]thiazole (DPTTZ). The framework exhibits through-space intervalence charge transfer (IVCT) arise from cofacially arranged DPTTZ linkers (Figure 1). The IVCT is elucidated computationally using time-dependent density functional theory (TD-DFT) methods. The computational study also exploits the distance-dependent through-space intervalence charge transfer (IVCT) in this system.

Here, I will present experimental observation of through-space intervalence charge transfer (IVCT) using redox-active organic linkers in the metal-organic framework and its computational understanding using TD-DFT. This interrogation of charge transfer mechanism and electrical conductivity in MOF provides a better understanding of conducting materials.

[1] Furukawa, H., Cordova, K. E., O'Keeffe, M., Yaghi, O. M. Science 2013, 341, 1230444.

[2] Sun, L.; Campbell, M. G.; Dincă, M. Angew. Chem. Int. Ed. 2016, 55, 3566.

[3] Hua, C. et al. J. Am. Chem. Soc. 2018, 140, 6622.

[4] Nath, A. et al. (Manuscript under preparation)

Molecular framework materials can combine the functional properties typical of the traditional inorganic solid state, such as magnetism, with the remarkable tunability and flexibility that arises from the incorporation of molecular components. They therefore offer the opportunity to discover unusual behaviour that arises from the coupling of these properties.

We have recently shown that thiocyanate (NCS–) based frameworks are a fruitful ground for the study of these novel properties, as thiocyanate can both facilitate strong magnetic coupling (TCW>100K, [1]) and create intense optically absorption in the visible region [2]. Despite this, the rich chemistry of metal thiocyanates remains unexplored compared to the equivalent metal formates, azides or hypophosphites.

Our investigations of the functional properties of metal thiocyanate frameworks began with the simplest examples: the layered binary thiocyanates, M(NCS)2. We demonstrated, through powder neutron diffraction studies, that this M(NCS)2 family possesses a wide variety of interesting magnetic phases. As part of this investigation we synthesised three new binary materials, M = Cu, Mn, Fe; and demonstrated that their magnetic interactions are significantly stronger than the previously studied exemplars (M=Co,Ni) increasing |TCW| by a factor of four.[1] Our results also uncovered that Cu(NCS)2 is a good example of a quasi-1D quantum Heisenberg antiferromagnet which a significantly reduced ordered moment in its ordered state.[3]

We have also investigated the family of CsM(NCS)3 materials which adopt the 'post-perovskite' structure.[4] The post-perovskite structure type is so-called as it occurs at pressures beyond the stability field of conventional perovskites (e.g. MgSiO3, CaIrO3, NaMnF3), but these molecular post-perovskites readily form in standard solution chemistry. We find that this family of materials shows significantly reduced ordering temperatures and adopt non-collinear magnetic structures that give rise to considerable magnetic hysteresis.[5]

[1] E. Bassey et al., Inorg. Chem. (2020).
[2] M. Cliffe et al., Chem. Sci., 10, 793 (2019).
[3] M. Cliffe et al., Phys. Rev. B, 97, 144421 (2018).
[4] M. Fleck, Acta Crystallogr. C60, i63 (2004).
[5] M. Geers et al., in prep.

Single-molecule magnets (SMMs) are molecular compounds possessing a magnetic bistability of their ground state, allowing them to maintain the direction of induced magnetization for a significant amount of time, after having first applied an external magnetic field [1]. Understanding the driving force behind good single-molecule magnet properties and developing improved rational synthesis design of them go hand in hand. This has been demonstrated in recent years, with record-breaking magnetic properties found in SMMs that utilizes a single Dy(III) centre in a highly axial ligand field [2-3]. A compound designed with this in mind is the pentacoordinate [Dy(Mes*O)₂(THF)₂Br]3THF (Mes*: 2,4,6-tri-tert-buylphenyl, THF: Tetrahydrofuran, DyBrTHF), Figure 1 (left). In a recent study on this compound, the molecular environment was found to be critical for the magnetic properties [4].

One way of systematically changing the molecular environment is through induced hydrostatic pressure. The resulting structural changes can then be probed using X-ray diffraction (XRD), by utilizing a diamond-anvil cell (DAC). We have performed high-pressure single-crystal XRD at several pressure points up until 2.9(2) GPa, and analysed the ensuing structures. Looking at the first coordination sphere, we can investigate how the applied pressure alters the molecular environment of Dy, Figure 1 (middle). At the last two pressure points, a slight drop is noted for some of the Dy-O bonds.

The magnetic properties of SMMs are closely tied to their electronic structure, which can change when undergoing external pressure, as investigated earlier in our group [5]. This information can be accessed through theoretical ab initio calculations, done here using CASSCF+NEVPT2 in ORCA. The found NEVPT2 energies of the Kramers doublets at varying pressure reveal a significant change in the energy levels, Figure 1 (right), perhaps due to the pressure-induced alteration of the ligand field.

Oxide ion conductors, used in separation membranes, electrolysers and fuel cells, are typically three dimensional isotropic materials, providing fast ion diffusion pathways. These are typically oxides that have been aliovalently substituted to enhance the concentration of mobile defects, most notably oxygen vacancies. One alternative strategy is to consider materials with anisotropic conduction pathways, and with excess oxygen, accomodated as interstitials. Based on this strategy we have recently investigated a series of oxides, including CeTaO4.17, CeNbO4+d (d = 0, 0.08, 0.25) and developed from this our interest in the structurally related La(Nb,W)O4+d compositions.

Each of these oxidised Ce based phases are known to adopt either a commensurate of incommensurate modulated structure, depending on the level of excess oxygen accommodated [1,2], but from a device perspective performed poorly as the Ce³⁺/Ce⁴⁺ ratio introduced undesriable electronic conductivity. In an effort to maintain the modulated structure(s), suppress electronic charge transport and enhance oxygen transport, we have targeted the LaNb_1-xW_xO_4+d sereis of materials. Our studies have developed the solid solution series phase chemistry and from application of X-ray, neutron and electron diffraction techniques, identified a sequence of modulated monoclinic and tetragonal phases. We have probed the ion transport of a select number of these phases, proving their capability as oxide ion conductors. We highlight the local structrure and variation in the coordination environments that facilitate the fast ion transport, offering routes to optimise and develop new functional oxides.

The collective motion of electrons has always been a fascinating topic in condensed matter physics. In charge density wave (CDW) systems, transport measurements were the first to provide a clear signature of the collective motion of condensed electrons. A non-linear conductivity is observed above a threshold current IT and is attributed to depinning of the CDW on impurities. An excess current then arises as well as a broad band noise and current oscillations. Although the electron density modulation involved in CDWs is very small, x-ray diffraction provides information about the structure of the CDW as it is associated with a periodic lattice distortion. We will show here how state-of-the art x-ray diffraction techniques - coherent and nanoprobe XRD - can reveal the different steps of CDW deformations, from pinning to sliding, in systems of increasing dimensions.

The structures of the rare earth metal polychalcogenides REX_2–δ (RE = La-Nd, Sm; Gd-Lu; X = S, Se, Te; 0 ≤ δ ≤ 0.2) attracted some attention due to their distorted square planar chalcogenide layer and the motives observed within these layers. All structures share a common structural motif of an alternating stacking of puckered [REX] and planar [X] layers (Figure 1a) and are closely related to the ZrSSi structure (space group P4/nmm), which is regarded as their common aristotype [1]. For electronic reasons, the planar [X] layer shows distortions from a perfect square net, forming dianions X₂^2– for the non-deficient REX₂. By reducing the chalcogenide content vacancies are observed within the planar layer, resulting in different superstructures for the REX_2–δ compounds depending on the vacancy concentration. For the sulfides and selenides this results in additional X^2– anions along vacancies to maintain a charge balanced layer. The tellurides, however, show different ordering patterns in the planar [Te] layer for the non-deficient RETe₂ compounds, but also a tendency to form larger anionic fragments for the deficient RETe_2–δ compounds, as seen for the commensurate structure of GdTe_1.8, e.g. [2].

LaTe_1.94 and LaTe_1.82 are two examples of different incommensurate crystal structures for RETe_2–δ compounds, separated by the number of vacancies in the planar [Te] layer [3, 4]. Both compounds share an average tetragonal unit cell with a ≈ 4.50 Å and c ≈ 9.17 Å, based on the structure of their aristotype (Figure 1a). The major difference of these compounds are their respective q vectors, which are compatible with tetragonal symmetry for LaTe_1.94, but indicate a loss of the fourfold rotational axis for LaTe_1.82, ending up in an orthorhombic superspace group. The [Te] layer of LaTe_1.94 is mainly composed of single vacancies (point defects), isolated Te^2– anions and Te₂^2– anions. LaTe_1.82 is more Te deficient and features adjacent vacancies in addition to Te₃^4– anions, to compensate for the missing charges (Figure 1b). To evaluate the formation of possible larger anionic fragments, like a bent Te₃^2– anion and the influence of additional vacancies to the structure, DFT based ELI-D real space analysis of approximant structures were performed (Figure 1c).

Figure 1. a) Average structure of LaTe_1.82; b) section of the modulated [Te] layer of LaTe_1.82; c) orthoslices of ELI-D of the Te layer of LaTe_1.82 with isocontour lines based on a commensurate approximant.

[1] Doert, T. & Müller, C. J. (2016). Reference Module in Chemistry, Molecular Sciences and Chemical Engineering, Elsevier.

[2] Poddig, H., Donath, T., Gebauer, P., Finzel, K., Kohout, M., Wu, Y., Schmidt, P. & Doert, T. (2018). Z. Anorg. Allg. Chem. 644, 1886–1896.

[3] Poddig, H., Finzel, K., Doert, T. (2020) Acta Crystallogr. Sect. C 76, 530–540.

[4] Poddig, H., Doert, T. (2020), Acta Crystallogr. Sect. B, 76, 1092–1099.

Ni-Mn-Ga-based Heusler alloys are broadly studied for their magnetic shape memory (MSM) functionality originating from coupling between ferroelastic and ferromagnetic orders. The ferroelastic order is established after martensitic transformation. Formed ferroelastic domains with different orientation are separated by twin boundaries. In modulated phases, these boundaries are extremely mobile and can be manipulated by magnetic field. Thanks to these, the single crystals of five-layered modulated 10M martensite of Ni-Mn-Ga exhibits magnetically induced reorientation (MIR) of ferroelastic (twin) domains in a moderate field of the order of 0.1 T [1, 2]. This results in 6 % magnetic field induced strain (MFIS) down to liquid helium temperature [3]. Such unique behaviour makes the 10M martensite a perfect candidate for applications in actuators, sensors and energy harvesters.

The ferroelastic microstructure represents a challenge for proper determination of martensite phase structure. Due to the modulated nature together with complex hierarchical twinning (compound and type I and II a/c twins; and non-conventional twins) [4, 5], the structure of the 10M martensite has not yet been completely solved. There is even an ongoing discussion about the nature of the modulation where two main concepts are considered: i) general crystallographic wave modulation approach, and ii) nanotwinning. As the structural modulation seems to be the critical factor for the extremely high twin boundary mobility [5], the problem is pressing.

Using the X-ray and neutron diffraction, we investigated on the character and temperature evolution of 10M martensite phase. We found transition from commensurate to incommensurate 10M modulated structure in Ni₅₀Mn₂₇Ga₂₂Fe₁ single crystal [6]. The modulation vector gradually increases upon cooling from commensurate q = (2/5) g₁₁₀, where g₁₁₀ is the reciprocal lattice vector, to incommensurate with q up to pseudo-commensurate q = (3/7) g₁₁₀. Further cooling results in transition to 14M with q = 2/7 g₁₁₀. Upon heating, reverse changes of the commensurate-incommensurate transition are observed with a thermal hysteresis of ≈ 60 K. We detected the same hysteretic behaviour in the electrical resistivity and the effective elastic modulus. Scanning electron microscopy showed that the changes are accompanied by the refinement of the a/b laminate.

Furthermore, we observed continuous modulation changes within the 10M martensite of wide range of Ni-Mn-Ga(-Fe) compositions that undergo the Austenite → 10M → 14M martensite transition sequence. Based on these observations, we suggest that the commensurate state is a metastable form of 10M martensite. Upon cooling, this phase evolves through nanotwinning into a more irregular and more stable incommensurate structure, further supported by recent high-resolution TEM observation [7].

[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.

The tungsten bronzes are low-dimensional transition metal oxides of great interest for their electronic instabilities. They show exotic physical properties such as superconductivity and charge density wave (CDW) phases. An important subfamily is (PO₂)₄(WO₃)_2m, which is interesting for its optical/magnetic behaviours, where the band filling and CDW phases coupled in different way with the lattice. These properties can be tuned by m, the thickness of the perovskite-like WO₆ –octahedra block¹.

To understand the electronic instabilities, correlated to the nesting properties of the Fermi surface and the consequent CDW phases, we used the combination of two techniques: diffuse scattering (DS) and inelastic x-ray scattering (IXS). This allows rapid identification of the nature of diffuse features in the patterns and the study of the lattice dynamics. Three different members are chosen in order to show the evolution of the behaviour in the family. In this context, we will focus on the lattice dynamics and framework instability. The first member, m=2, presents a quasi-1D instability given by the WO₃-octahedra zig-zag chains, which are isolated by the phosphates. A CDW phase is found, T_C=270K, and it is linked to a rigid-body motion. Different behaviour can be found in the members m=6 and 8, where the instability is found in the WO₃ slabs, realised as correlated displacements of tungsten atoms along the octahedral 4-fold axis direction. The three members show different diffuse patterns, figure 1. The results are linked to the lattice dynamics behaviour, which present a Kohn anomaly above the transition temperature, however as predicted from the diffuse results, has a different Q- and temperature-dependence in each member.

[1] P. Roussel et al., Acta Cryst. B 57 (2001) 603-632

The novel aperiodic titanate Ba₁₀Y₆Ti₄O₂₇ has a thermal conductivity that equals the lowest reported for an oxide at room temperature. All of the atomic sites are described by crenel function occupancy modulations. The resulting localisation of lattice vibrations suppresses phonon transport of heat. Thus Ba₁₀Y₆Ti₄O₂₇ represensts a new lead material for low thermal conductivity oxides, the possibility of using the structural description to slect other new leads will be explored.

The word “chiral”, introduced by Lord Kelvin in 1904, refers to an object whose image in a plane mirror does not coincide with itself [1]. One intuitive example is the left and right hands, which are mirror images of each other but are not superimposable. The two forms of a chiral object are called enantiomorphs or enantiomers for molecules. The chirality is a key property which is found in all branches of science, from biology to physics and at different scales, from microscopic to macroscopic objects. For instance, in biology, the notion of chirality is crucial for living organisms and plays a critical role in molecular recognition [2]. In parallel, in fundamental physics, the chirality is also an important property, as shown by the example of the weak interaction, not invariant under mirror symmetry [3], which only interacts with left-chiral fermions or right-chiral anti-fermions. The chirality can also be found in solid state physics, in crystallography where it refers to the concept of spatial inversion symmetry rather than mirror symmetry, or again in magnetism, where it refers to the sense of rotation of the spins on oriented loops [4].

In this talk, I will focus on the concept of chirality in magnetic ordered systems. I will present the archetype chiral magnetic compound, Ba₃NbFe₃Si₂O₁₄, which hosts three different types of chirality. This system belongs to the family of langasite materials providing interesting geometrically frustrated spin lattices. It crystallizes in the non-centrosymmetric P321 space group and displays a structural chirality. The magnetic Fe³⁺ ions form an original triangular network in the (a,b) planes, stacked along the c-axis (see Figure 1). Below T_N ~ 27 K, the system orders magnetically with a 120° spins structure within each triangle, in the (a,b) planes, and presents a helical modulation along the perpendicular direction, i.e. the c-axis, with a period of ~ 7 lattice parameters (see Figure 1) [5]. Surprisingly, this magnetic ground state displays a unique sense of rotation of the spins within the triangles (triangular chirality) as well as a unique sense of rotation of the spins along the helices (helical chirality). This multi-chiral magnetic ground state is correlated to the structural chirality through a twist of the inter-plane exchange interactions (see Figure 1) [5-7]. I will present the scientific arguments that led to the discovery of such complex multi-chiral magnetic structure and the consequences on its physical properties. I will conclude by presenting our last results focusing on the critical regime and the nature of the phase transition toward this peculiar multi-chiral magnetic order.

[1] Lord Kelvin, (1904). Baltimore Lectures. London: C. J. Clay and Sons 619.

[2] Inaki, M., Liu, J., & Matsuno, K., (2016). Phil. Trans. R. Soc. B 371, 20150403.

[3] Lee, T. D. & Yang, C. N., (1956). Phys. Rev. 104, 254. Lee, T. D., Oehme, R. & Yang, C. N., (1957). Phys. Rev. 106, 340.

[4] Simonet, V., Loire, M. & Ballou, R., (2012). Eur. Phys. J. Spec. Top. 213, 5.

[5] Marty, K., Simonet, V., Ressouche, E., Ballou, R., Lejay, P. & Bordet, P., (2008). Phys. Rev. Lett. 101, 247201.

[6] Loire, M., Simonet, V., Petit, S., K. Marty, Bordet, P., Lejay, P., Ollivier, J., Enderle, M., Steffens, P., Ressouche, E., Zorko, A. & Ballou, R., (2011). Phys. Rev. Lett. 106, 207201.

[7] Chaix, L., Ballou, R., Cano, A., Petit, S., de Brion, S., Ollivier, J., Regnault, L.-P., Ressouche, E., Constable, E., Colin, C. V., Zorko, A., Scagnoli, V., Balay, J., Lejay, P., & Simonet, V., (2016). Phys. Rev. B 93, 214419.

In metallic heavy fermion materials the magnetic ground state is often a spin density wave (SDW) phase in which the magnetization vary periodically with a period that is usually incommensurate with the parent structure lattice. These phases are associated to the itinerant character of the f-electron present in the system and are intimately related to the electronic structure near the fermi energy.

This is the case in the tetragonal heavy-fermion compound CeAuSb₂ which shows the development of a SDW phase below T_N~6.5 K with a propagation vector k₁ = (0.136, 0.136, 0.5) [1-2]. This phase is very sensitive to external stimuli and, indeed, the systems shows two metamagnetic phase transitions with magnetic field applied along the [001] direction [1-2]. An additional parameter which can tune the magnetic ground state is the application of a uniaxial stress, for example along the [010] direction. Extensive transport and thermodynamic measurements [2, 3, 4] indicate a sudden and anisotropic jump of the resistivity at an induced strain along the axis of compression of 0.5% indicating a first order transition.

In this talk we present single crystal time of flight neutron diffraction data collected under the application of a [010] uniaxial stress to characterize the magnetic phases of CeAuSb₂. The neutron data indicate a change of the propagation vector from k₁ at low stress to k₂ = (0, 0.25, 0.5) at high stress. Even with the geometrical constrains imposed from the experiment sample environment, which allows to collect only a limited number of magnetic reflections, we will show that it is possible to determine and refine the magnetic structure with the support of group theory calculations and magnetic symmetry analysis. The commensurate nature of the propagation vector is attributed to the presence of a lock in invariant in the free energy and we will show that the magnetic ground state under compressive stress is characterized by the presence of two primary order parameters related to different irreducible representations of the parent structure.

[1] Marcus, G. G., Kim, D.-J., Tutmaher, J. A., Rodriguez-Rivera, J. A., Birk, J. O., Niedermeyer, C., Lee, H., Fisk, Z., Brohol, C. L. (2018). Phys. Rev. Lett. 120, 097201

[2] Zhao, L., Yelland, E. A., Bruin, J. A. N., Sheikin, I., Canfield, P. C., Fritsch, V., Sakai, H., Mackenzie, A. P., Hicks, C. W. (2016). Phys. Rev. B 93, 195124.

[3] Park, J., Sakai, H., Erten, O., Mackenzie A. P., Hicks, C. W. (2018). Phys. Rev. B 97, 024411 [4] Park, J., Sakai, H., Mackenzie A. P., Hicks, C. W. (2018). Phys. Rev. B 98, 024426.

[4] Park, J., Sakai, H., Mackenzie A. P., Hicks, C. W. (2018). Phys. Rev. B 98, 024426.

Magnetic interactions are the fundamental components for the fascinating variety of complex magnetic structures and properties found in many functional materials. Identifying, understanding, and finally predicting these interactions is an essential step towards their utilization in novel devices. One of these basic interactions is the Dzyaloshinskii-Moriya interaction (DMI) – an antisymmetric exchange coupling favouring a perpendicular arrangement of magnetic moments, and thus a canting in otherwise collinear structures [1,2]. The DMI, originally introduced in the late 1950s to explain ‘weak ferromagnets’ (not perfectly collinear antiferromagnets), regained the interest in current condensed matter research as it was found to be the driving force to stabilize various novel topological noncollinear magnetic structures, such as spin spirals [3], magnetic skyrmions [4], magnetic soliton lattices [5] and others. In particular for spintronic applications, the DMI shows promising characteristics towards the development of next-generation devices [6]. Although the magnitude of the DMI-induced canting is usually small, the direction can have a fundamental impact on the spin chirality and the resulting magnetic and multiferroic properties [7]. Here, we present polarized neutron diffraction (PND) as an efficient technique for the determination of the absolute direction of the DMI in weak ferromagnetic materials, as recently established by us [8].

We provide the basic formalism for a symmetry analysis of the DMI in crystal structures and show how to relate the measured PND data with the absolute DMI direction. We exemplify this approach in weak ferromagnetic MnCO₃ and identify the magnetic moment configurations for a positive or negative sign of the DMI with an applied magnetic field as shown in Fig. 1. Using PND [9], we can distinguish even from the measurement of a single suitable Bragg reflection between the two configurations and unambiguously reveal a negative DMI sign in MnCO₃. This is in agreement with previous results obtained by resonant magnetic X-ray scattering and thus, validates the method [10]. We demonstrate the generality of our method by providing further examples of topical magnetic materials with different symmetries and support our findings with ab-initio calculations, which reproduce the experimental results.

Figure 1. The local environment of the z=0 manganese atom in the hexagonal unit cell of MnCO₃. The six nearest-neighbour manganese atoms of the other magnetic sublattice are shown as light and dark blue spheres located above and below the central atom, respectively. The oxygen atoms between these manganese layers are shown as small yellow spheres. Panels (a) and (b) show the two possible magnetic moment configurations stabilized dependent on the sign of the D_z DMI component by applying an external magnetic field along the [110] direction aligning the weak ferromagnetic moment.

[1] V. E. Dzyaloshinskii, Sov. Phys. - JETP 5(6), 1259 (1957)

[2] T. Moriya, Phys. Rev. 120(1), 91 (1960)

[3] M. Bode et al., Nature 447, 190 (2007)

[4] S. Heinze et al., Nat. Phys. 7, 713 (2011)

[5] Y. Togawa et al., Phys. Rev. Lett. 108, 107202 (2012)

[6] S. S. P. Parkin et al., Science 320, 190 (2008)

[7] J. Cho et al., J. Phys. D: Appl. Phys. 50, 425004 (2017)

[8] H. Thoma et al., Phys. Rev. X 11, 011060 (2021)

[9] H. Thoma et al., J. Appl. Crystallogr. 51, 17 (2018)

[10] V. E. Dmitrienko et al., Nat. Phys. 10, 202 (2014)

Ca₂RuO₄ (CRO), the close neighbour of the famous superconductor Sr₂RuO₄ displays surprisingly different behaviour to its neighbour, exhibiting insulating behaviour below an irreversible metal-insulator transition at T_MI = 357K. In the insulating state CRO displays orbital ordering at T_OO = 260K and antiferromagnetic ordering below T_N = 110K. This material has been extensively investigated but still questions remain regarding the nature of the insulating state and whether Mott gaps are opened only on certain orbitals, or whether the insulating state is a result of purely structural change. While recent publications have tended towards the latter of these possibilities, previous results observing varying orbital concentrations with temperature have not been explained. Here we will show new resonant elastic x-ray scattering (REXS) results from the Ruthenium absorption edge made on the synchrotron beamline I16 at Diamond. The resonant spectra provide a unique way of looking at the ordered magnetic and orbital structure of this material and we will present a systematic approach to understanding the different contributions to these signals.

Binary rare-earth intermetallic compounds of R₇Rh₃ type attract possess complex magnetic phase diagrams and rich variety of magnetic structure transitions. In particular, three temperature induced magnetic phase transitions were observed at T_N = 32 K, T_t1 = 21 K, and T_t2 = 9 K [1, 2]. In this work, a comprehensive study of the low-temperature magnetic state of Ho₇Rh₃ was carried out using neutron diffraction and nonlinear AC magnetic susceptibility.

Analysis of the neutron diffraction data and the temperature dependence of the harmonics χ_nω'(T) and χ_nω''(T) (n = 1, 2, 3) (Fig. 1) showed that the magnetic phase transition at a temperature T_N = 32 K is associated with emergence of an incommensurate magnetic structure of spin density wave type described by the magnetic superspace group Cmc2₁1'(00g)0sss. Upon further cooling below the temperature T_t1 ~ 21 K, a "squaring-up" process begins reflecting evolution of the amplitude modulated incommensurate magnetic structure towards a rectangular structure of the "antiphase domains" type. At T<T_t2 ~ 9 K, the magnetic structure can be described by the magnetic supersymmetry groups Cm'c2₁'(00g)ss0 or Cmc'2₁'(00g)000, which are subgroups of index i = 2 of the Cmc2₁1'(00g)0sss magnetic superspace group. Symmetry breaking associated with {1’|0 0 0 1/2} operation lost at the transition allows the emergence of a spontaneous magnetization confined in the basal plane of the hexagonal structure Ho₇Rh₃ while magnetic structure keeps its incommensurate character. Measurements of the linear and nonlinear AC magnetic susceptibility revealed that emergence of the weak spontaneous magnetization in the sample are accompanied by pronounced anomalies in the temperature dependencies of the 2nd and 3rd harmonics of the AC susceptibility ascribed to a symmetry breaking due to the loss of the time inversion symmetry {1’|0 0 0 1/2}.

[1] Tsutaoka, T., et al, (2003). Physica B. 327, 352-356.

[2] Tsutaoka, T., et al, (2016). J. of Alloys and Compounds. 654, 126-132.

Detailed studies of electron density changes in a mineral called langbeinite K₂Mg₂(SO₄)₃ under pressure have been performed. Single crystal X-ray data for this mineral under pressure (1GPa) were collected at the 13-BM-C beamline at the Advanced Photo Source (Argonne National Laboratory, USA). Additionally, complementary experiments at ambient conditions were performed on an in-house diffractometers. Experimental results were complemented by theoretical calculations within the pressure range up to 40 GPa.

From the point of view of mineralogical processes taking part in the Earth mantle (and the mantles of other even extraterrestrial planets), establishing detailed changes of electron density in minerals under pressure is absolutely crucial to understand the nature and mechanisms of mineralogical processes. Combining both experimental charge density studies and high pressure investigations is still a real challenge. This work is our continuation of our previous feasibility studies on experimental quantitative electron density investigations of electron density in grossular under 1GPa pressure [1].

Answering the questions how electron density distribution in langbeinite is affected by increasing pressure is obviously the main topic of this work. However there are also some other issues which we would like to address. Are there any differences between experimental and theoretical charge density distributions obtained on the basis of experimental data and theoretical dynamic structure factors? Are there any significant differences in properties of charge density distributions obtained for complete and incomplete high resolution X-ray diffraction data sets? Are there any differences in charge density distributions obtained for X-ray data collected with two different wavelengths of X-ray radiation? Should the data be absolutely complete to obtain reasonable experimental charge density distribution? When experimental data are impossible to be collected, is it reasonable to use theoretically calculated dynamic structure factors instead and refine theoretical models of electron density?

Langbeinite crystalizes in the cubic P2₁3 space group. Its structure is composed of SO₄ tetrahedra and MgO₆ octahedra. Potassium cations which are placed in the voids between these polyhedra are surrounded by oxygen anions. Unfortunately due to significant deformation, one cannot say that KO₁₂ is a regular icosahedron. Although mentioned polyhedra seem to completely fill in the space, this schematic way of presentation is not the best one when topology of electron density distribution must be described.

Investigating changes of electron density as a function of pressure, we are going to compare electron density properties at BCPs, integrated atomic basins, changes of thermal ellipsoids. Obviously, raising pressure will cause shrinking of the unit cell and consequently, changes of electron density distribution. However, the question is how exactly such changes will manifest.

No doubt that polyhedra commonly used in mineralogy and crystallography are not useful representation of electron density as they neither have full representation of electron density of the central ion nor any of the corners ions. So from time to time returns an old question: how big are atoms in crystals [2]. Here we will answer this question and the other ones already mentioned above at the level of quantitative electron density distributions in our model mineral.

[1] Gajda, R., Stachowicz, M., Makal, A., Sutuła, S., Parafiniuk, J., Fertey, P. & Woźniak, K. (2020). IUCrJ. 7, 383-392.

[2] Brown, I. D. (2017). Struct. Chem. 28, 1377-1387.

Keywords: high pressure; electron density; theoretical structure factors

A quantitative experimental charge density study was undertaken for the double antiperovskite mineral – sulphohalite [Na₆(SO₄)₂FCl]. High-resolution X-ray diffraction data was collected employing AgKα radiation (λ = 0.56087 Å) to a resolution of 0.3941 Å at 100K. Electron density (ED) distribution – ρ(r) was modelled, in compliance with the Hansen-Coppens formalism[1], by consecutive least-square multipolar refinements. Based on such experimental distribution of charge, QTAIM topological analysis[2] was undertaken. Full-volume property integration over delineated atomic basins (AB’s) yielded their appertaining charges [Q_AB-Cl = -0.836e^-; Q_AB-S = 03.168e^-; Q_AB-Na = 0.910e^-; Q_AB-F = -1.334e^-; and Q_AB-O = -1.227e^-] and volumes [V_AB-Cl = 38.920ų; V_AB-S = 5.656ų; V_AB-Na = 7.931ų; V_AB-F = 14.178 ų and V_AB-O = 17.416 ų]. The percentage of unaccounted electrons and volume per unit cell was respectively 0.010% and 0.406%. Within the uncertainty range of performed numerical integration, such percentages can be unheeded. A total of 6·BCP’s [∇²ρ(r_Cl···S) = 0.120e^-·Å^-5; ∇²ρ(r_Cl···Na) = 0.575e^-·Å^-5; ∇²ρ(r_S-O) = -31.00e^-·Å^-5; ∇²ρ(r_Na···O) = 1.931e^- ·Å^-5; ∇²ρ(r_Na···F) = 3.022e^-·Å^-5 and ∇²ρ(r_F···O) = 0.868e^-·Å^-5], 5·RCP’s [∇²ρ(r_I) = 0.912e^-·Å^-5; ∇²ρ(r_II) = 0.332e^-·Å^-5 and ∇²ρ(r_III,IV,V) = 0.201e^-·Å^-5] and 4·CCP’s [∇²ρ(r_I,II) = 0.514e^-·Å^-5 and ∇²ρ(r_III,IV) = 0.401e^-·Å^-5] were identified (Figure 1). Hence, Morse’s ‘characteristic set’ condition was met[3]. The study of primary bundles (PB’s), as proposed by Pendás[4], revealed the interconnection between AB’s and CP’s onto basins of attraction or basins of repulsion. The nature of interatomic interactions was assessed through the dichotomous classification[3]. The S–O contact was acknowledged as a covalent with a shared-shell. The remaining contacts were characterized as non-covalent closed-shell (Cl···Na, Na···O and Na···F) or weak van der Waals closed-shell (Cl···S and F···O).

Modern approaches of X-ray diffraction allow for detailed quantitative studies of electron density in crystals of minerals. They can be combined with high pressure studies [1] as we demonstrate in this work for model zeolite mineral hsianqhualite, Ca₃Li₂(Be₃Si₃O₁₂)F₂.

At the level of electron density analysis first order configurational components in crystal structure description (Fig. 1a) were replaced by Bader’s atomic basins [2] which quantitatively characterise electron density of particular ions in mineral structures as well as precisely defined space, they occupy (Fig 1b). Their anisotropic and highly non-spherical shape reflects interatomic interactions and is sensitive to applied pressure. According to our studies the charge of ions in the crystal lattice differ from the formal, integer values and when external pressure is applied a redistribution of charge among ions takes place. This redistribution changes the size and shape, mostly at the edges of ionic basins in nonbonding fragments (Fig. 1c, d).

Negative compressibility of the F ion was observed. It was caused by the flow of electrons increasing the total negative charge and, consequently, increasing the volume of F ion at 1.9 GPa pressure (Fig 1d). Also inside of atomic basins of atoms electron density redistributes notably due to pressure.

The quantitative characterization of minerals under high pressure at the subatomic level of electron density, rise possibilities to better understand the nature of mineralogical process, phase transitions and formation of new phases and also to study plastic deformations of minerals using diamond anvil cells.

Zeolites are commonly used as ion-exchange materials for the remediation of nuclear waste; however, they have certain drawbacks. Unlike zeolites which contain SiO₄ and AlO₄ tetrahedra, microporous Ti-silicates can contain SiO₄ tetrahedra and TiO₆ octahedra and therefore structures are possible which have no traditional aluminosilicate analogues ^[1]. Microporous Ti-silicates such as sitinakite KNa₂Ti₄Si₂O₁₃(OH)·4H₂O and the synthetic niobium doped analogue are used for the removal of Cs⁺ and Sr²⁺ from nuclear waste ^[2,3]. The work presented here will focus on the structures and thermal behaviour of the ion-exchanged Ti- and Zr-silicates. A clear understanding of both is fundamental in determining if these materials have potential as ion-exchangers within the nuclear industry.

Umbite is a naturally occurring small pore microporous Zr- silicate, found in northern Russia and synthetic analogues, K₂ZrSi₃O₉·H₂O, can be prepared in the laboratory ^[4]. Ion-exchange studies here have shown that umbite has a preference for common radionuclides, such as Cs⁺ and Sr²⁺and Ce⁴⁺ (as a surrogate for Pu), even in the presence of competing ions. In-situ studies show that these materials behave differently with temperature, indicating that the nature and location of the charge balancing cation plays an important part in determining which high temperature phases are formed and the phases formed do not fit previously reported structures.

Natisite is another material which has interesting ion-exchange chemistry and is a layered Ti-silicate with the formula Na₂TiSiO₅ ^[6]_. The structure consists of square pyramidal titanium, with the sodium cations located between the layers. This coordination environment is highly unusual for Ti. Inclusion of zirconium or vanadium in the framework has a considerable effect on the ion-exchange properties, with changes in the exchange capacity and the rate of uptake for certain ions of interest.

A combination of techniques to probe long and short-range order (PDF and XAS) have been used to understand the ion-exchange and thermal behaviour of these materials.

References:

1) P. A. Wright, Microporous Framework Solids, The Royal Society of Chemistry, Cambridge, 2008. 2) D. M. Poojary, et al., Chem. Mater., 6, 2364 (1994). 3) A. Tripathi, et al., J. Solid State Chem., 175, 72 (2003). 4) D. M. Poojary, et al., Inorg. Chem., 36, 3072 (1997). 5) A. Ferreira, et al., J. Solid State Chem., 183, 3067 (2010). 6) D.G. Medvedev et al., Chem. Mater., 16, 3659 (2004).

The synthesis of multifunctional materials is a hot focus of research in materials science. In this respect, the synthesis of complexes based on the combination of organic-inorganic building blocks provides a promising approach in the design of systems with tuneable properties. In this communication we will present the properties of a new compound based on quinuclidine as the organic cation and FeCl₄ as the inorganic anion, with the formula (quinuclidine)[FeCl₄]. Similar compounds derived of this heterocyclic cation have been found to present interesting ferroelectric properties.[1] In this context, the multifunctional behaviour of this novel molecular crystal is related to the electronic structure of the 3d⁵ configuration of the Fe(III) ions together with the ability of the counter-ions to change of orientation or even become disordered as a function of temperature.

The structural characterization of (quinuclidine)[FeCl₄] compound shows two phase transitions. The first one, detected in the range from 100 to 300 K, was resolved by single-crystal X-Ray and neutron diffraction. At 300 K, the compound presents the orthorhombic space group Pbc2₁. At 100 K, the space group is Pbca, with a doubling of the a-axis, related to the rotation of the cations: two different orientations of the counterion are observed in the low temperature phase along the a direction, contrary to the high temperature phase, where it appears only one orientation.

Moreover, this compound presents long-range magnetic order below 3 K. The magnetic structure was solved using single-crystal and powder neutron diffraction data from D19 and D1B instruments (ILL, France), respectively. Our best model was found on the Shubnikov magnetic space group P2₁’2₁’2_1. Although the refined model present an antiferromagnetic structure, based on the symmetry analysis of the P2₁’2₁’2₁ Shubnikov group, a ferromagnetic component along the c direction is allowed. However, the refinement of this ferromagnetic component is beyond the precision of our measurements. Nevertheless, this can be fixed to the values derived from the macroscopic magnetometry measurements (SQUID). In order to provide a complete model these values were included in the magnetic model and fixed during the refinements.

At temperatures higher than R.T, there is a second structural phase transition which produces an important modification of the electric behaviour, as it has been reported on similar compounds.[1] The dielectric permittivity data collected shows a sharp phase transition around 390 K (also observed in DSC measurements). The value of the permittivity increases drastically with the increase of the temperature, reaching a maxima of 10⁵ at 390 K (measured at 1 kHz). This value is notable larger than similar compounds of this family.[1] This interesting behaviour could be of interest for electrochemical applications.

[1] (a) Jun Harada et al., Nat Chem, 2016 Oct; 8(10):946-52. (b) You-Meng You et al., Nat Commun. 2017, 8:14934. c) Ting Fang et al., Z. Anorg. Allg. Chem. 2019, 645, 3–7. d) Guang-Meng Fan et al., CrystEngComm, 2018,20, 7058-7061.

Non-covalent intermolecular interactions polarize a drug molecule in the biological environment to prepare it for the recognition and binding process with a related enzyme. In a crystal structure of the same drug molecule, the crystal packing is defined by the same kind of non-covalent interactions. This means that in both a biological as well as a crystalline environment, the small molecule will conformationally adapt its shape to the prevailing intermolecular binding forces, so that the resulting bound state reflects both its inherent flexibility and the environment. Electrostatic complementarity between an enzyme binding site and an active molecule is an aspect that goes beyond geometry and molecular conformation since the electrostatic potential is inherently related to the electron density distribution. We ask to which extent small-molecule crystal structures can be used to predict the conformation and interaction density of the same molecule in the enzyme.

The first compound class investigated is related to loxistatin acid E64c. These compounds are cysteine protease inhibitors, and the active site is an electrophilic epoxide ring.[1] It took us many years to solve the small-molecule crystal structure of E64c,[2] and experimental electron-density studies were only possible for related model compounds.[3] Recently, however, we were able to perform a full quantum-crystallographic, molecular-dynamics and QM/MM study of the active site of E64c co-crystallizing in a system that closely resembles the binding situation of E64c in the cysteine protease cathepsin B.[4]

The second compound class investigated refers to ibuprofen derivatives. We used the umpolung principle to tune the properties of ibuprofen by carbon-silicon exchange, which in turn impacts on the electrostatic complementarity relationships when ibuprofen binds to cyclooxygenases.[5] Again, we investigated the enzyme and crystal environmental effects on ibuprofen and sila-ibuprofen by quantum crystallography, molecular dynamics and QM/MM calculations.[6]

Every low-temperature high-resolution single-crystal X-ray diffraction experiment utilized in this study was conducted at a synchrotron beamline at either DESY, APS or SPring-8. Without access to large infrastructure, such studies on weakly scattering pharmaceutically active compounds would not be possible. I will therefore not only report the biochemically relevant results, but also the importance of synchrotron experiments for our field.

[1] Mladenovic, M., Ansorg, K., Fink, R. F., Thiel, W., Schirmeister, T. & Engels, B. (2008). J. Phys. Chem. B, 112, 11798.
[2] Shi, M. W., Sobolev, A. N., Schirmeister, T., Engels, B., Schmidt, T. C., Luger, P., Mebs, S., Dittrich, B., Chen, Y.-S., Bąk, J. M., Jayatilaka, D., Bond, C. S., Turner, M. J., Stewart, S. G., Spackman, M. A. & Grabowsky, S. (2015). New J. Chem. 39, 1628.
[3] Grabowsky, S., Schirmeister, T., Paulmann, C., Pfeuffer, T. & Luger, P. (2011). J. Org. Chem. 76, 1305.
[4] Kleemiss, F., Wieduwilt, E. K., Hupf, E., Shi, M. W., Stewart, S. G., Jayatilaka, D., Turner, M. J., Sugimoto, K., Nishibori, E., Schirmeister, T., Schmidt, T. C., Engels, B. & Grabowsky, S. (2021). Chem. Eur. J. 27, 3407.
[5] Kleemiss, F., Justies, A., Duvinage, D., Watermann, P., Ehrke, E., Sugimoto, K., Fugel, M., Malaspina, L. A., Dittmer, A., Kleemiss, T., Puylaert, P., King, N. R., Staubitz, A., Tzschentke, T. M., Dringen, R., Grabowsky, S. & Beckmann, J. (2020). J. Med. Chem. 63, 12614.
[6] Kleemiss, F., Duvinage, D., Puylaert, P., Fugel, M., Sugimoto, K., Beckmann, J. & Grabowsky, S. (2021). Acta Cryst. B, in preparation.

Visualizing on the atomic scale the full extent of the electronic and structural changes that are triggered by charge separation and subsequent charge transport is crucial for developing the rational design of novel sensitizers and catalysts. The rapid progress of ultrafast X-ray techniques, both at synchrotrons (100 ps) and at X-ray free electron laser facilities (sub-ps) have equipped the scientific community with novel analytical tools that are capable of delivering unique feedback with spin and elemental sensitivity about the highly-correlated nonadiabatic dynamics that follow photoabsorption. The present talk will review the technical state-of-the art and the ongoing developments that are currently taking place. The talk will also highlight several of the recent results that have been obtained for intramolecular and interfacial processes of relevance for the function and optimization of advanced materials.

Photoactive materials are among the most commonly researched and engineered functional materials, due to the multiplicity of applications they find in research and industry. Investigating of the dynamics of short-lived excited states in crystal structures allows us to extract information on how such materials could be designed on the molecular level in order to obtain desired properties. Coordination compounds containing group XI transition-metal atoms, such as copper (I), silver(I), or gold(I), are excellent examples of compounds with interesting and diverse photoactive properties, and thus were chosen for this study.

Time-resolved photocrystallographic methods allow us to investigate structural changes occurring due to formation of short-lived laser-induced excited-state species in crystals. For the following study, several coinage-metal mononuclear and multinuclear coordination compounds were examined using time-resolved X-ray-pump / laser-probe Laue experiments, conducted at the 14-ID-B BioCARS APS synchrotron beamline. The studied complexes include the literature-reported Ag(PP)(PS) (PP = 1,2-bis(diphenylphosphino)ethane, PS = 2-(diphenylphospaneyl)pyridine) and newly-synthesised Ag₂Cu₂(PS)₄ systems, both exhibiting bright luminescence in the solid state. The time-resolved data were processed with our home-made software and the photodifference maps were generated and analysed.

In order to comprehensively understand excitation-induced effects occurring in crystals, the abovementioned photocrystallographic measurements were supplemented with time-resolved luminescence spectroscopy experiments (355 nm excitation wavelength) and quantum computations yielding the nature of studied exited states and predicting the geometry changes (TDDFT and QM/MM methods) upon excitation. Results will be presented, and their accordance with photocrystallographic results assessed.

The authors thank NSC (2016/21/D/ST4/03753, 2014/15/D/ST4/02856) and WCSS (grant No. 285) in Poland, EU programme (POIG.02.01.00-14-122/09) and APS, USA (DOE: DE-AC02-06CH11357, NIH: R24GM111072) for financial support and access to facilities.

High-resolution X-ray diffraction experiments, theoretical calculations and atom-specific X-ray absorption experiments are applied to investigate two nickel complexes [Ni(II) (A) and Ni(III) (B)] (Figure 1a, 1b) with the non-innocent 3,6-dichlorobenzene-1,2-dithiolate ligand. Combining the techniques of metal K-, L-edge and sulphur K-edge X-ray absorption spectroscopy with high-resolution X-ray charge density studies, the experimental assessment of oxidation states of the central Ni atoms is studied and compared with theoretical predictions. Furthermore, the experimentally derived X-ray charge density (obtained via the multipole model) and the electron density from theoretical calculations are provided to further explore the contrast and contest of both approaches employed.

Figure 1. Compounds(a) (A), (b) (B), (c) Laplacian of electron density

The oxidation state of the central atom will be discussed [1] (Figure 1c).

[1] Machata, P., Herich, P., Lušpai K., Bučinský, L., Šoralová, S., Breza, M., Kožíšek, J. & Rapta, P. (2014). Rev. Sci. Instrum. 70, 3554.

Organometallics 33 (18), 4846.

Carbonyl…carbonyl interactions have been identified in biologically active systems such as small biomolecules, proteins or protein-ligand complexes. Their contribution into molecular assembly was proven to be comparable to moderate hydrogen bonds, thus they can be considered as organic synthons playing crucial role in determining three-dimensional crystal packing or even stabilizing the secondary structure motifs of proteins. In the literature one can find many examples of C=O…C=O interactions between the same molecules, however to the best of our knowledge, only one case where such a pattern was characterized between different molecules (urea and oxalic acid co-crystal) [1].

Here we report a series of co-crystals of urea and dicarboxylic acid, where antiparallel carbonyl…carbonyl motif [2] between heteromolecules is observed and acts as a “glue” between 2D layers built of strong hydrogen bonds. In order to get inside into the nature and mechanism of the synthons, experimental and theoretical electron density studies were engaged as well as ETS-NOCV method (Extended Transition State. Natural Orbitals for Chemical Valence) was applied [3]. NCI analysis [4] and interaction energies calculated with EP/MM (Exact Potential and Multipole Method) method [5] indicate a correlation between the strength of carbonyl interactions and the number of carbon atoms in the main chain of the acid molecules.

Literature:

[1] A. Krawczuk, M. Gryl, M. Pitak, K. Stadnicka Cryst. Growth Des. (2015), 15, 5578−5592.

[2] F.H. Allen, I.J. Bruno Acta Crystallogr., Sect. B: Struct. Sci. (2010), 66, 380−386.

[3] F. Sagan, M. P. Mitoraj J. Phys. Chem. A (2019), 123, 21, 4616–4622

[4] E.R Johnson, S. Keinan, P. Mori-Sánchez, J. Contreras-Garciá, A.J. Cohen, W. Yang, J. Am. Chem. Soc. (2010), 132, 6498−6506.

[5] A. Volkov, T. Koritsanszky, P. Coppens, Chemical Physics Letters (2004), 391 (1–3), 170–175.

Hirshfeld atom refinement (HAR)[1,2] is one of the most successful methods for the accurate determination of structural parameters for hydrogen atoms from X-ray diffraction data. It employs atomic scattering factors based on atomic densities obtained via Hirshfeld partition of theoretically determined electron density.

There are various ways of calculating the electron density with theoretical methods. For example, among others, we can independently change (1) a method of quantum chemistry (2) basis set and (3) a representation of molecular environment. This a leads to obvious question – which set of settings is the best for HAR refinement?

An another dimension was recently added to the space of settings by introducing generalization of HAR to other electron density partitions [3] (so called generalized atom refinement (GAR)). This makes the optimal choice of settings even more challenging.

Another factor further complicates the situation – computational cost of GAR. Usually unfavorable scaling of quantum chemical calculations with size of a system may lead to long refinement time for large molecules. While computational chemistry brings here some solutions, we still have to figure out how to handle trade-off between computational cost and accuracy of refinement.

In this contribution we will analyze effects of various settings of GAR on accuracy of the method (assessed by comparison to neutron data). We will also try to find optimal solution for performing accurate refinement with optimized computational cost.

[1] Jayatilaka, D. & Dittrich, B. (2008). Acta Cryst. A64, 383–393.

[2] Capelli, S. C., Bürgi, H.-B., Dittrich, B., Grabowsky, S. & Jayatilaka, D. (2014). IUCrJ, 1, 361–379

[3] Chodkiewicz, M. L., Woińska, M. & Woźniak, K. (2020). IUCrJ, 7, 1199–1215.

TBD

We have previously described FragLites, a library of small molecules that incorporate an anomalous scatterer for ready detection in X-ray crystallographic fragment screening. Here I will describe our findings as we have assessed their the potential of Fraglites for use in phase determination and to map protein structures for interaction hotspots.

Crystal harvesting remains the bottleneck in protein crystallography experiments and is the rate-limiting step for many structure determination, high-throughput screening and femtosecond crystallography studies. Huge progress has been made towards the automation of high-throughput crystallization, even for membrane proteins. Moreover, free electron lasers and fourth generation synchrotrons support extraordinarily rapid rates of data acquisitions and put further pressure on the crystal-harvesting step. Here [1], a simple solution is reported in which crystals can be acoustically harvested from slightly modified MiTeGen In Situ-1 crystallization plates. Acoustic harvesting uses the automated and keyboard driven acoustic droplet ejection (ADE) technology, in which an acoustic pulse ejects each crystal out of its crystallization well, through a short air column and directly onto a micro-mesh. Crystals can be individually harvested or can be serially combined with a chemical library such as a fragment library.

As crystallization plates are used in most automated high-throughput crystallization robots, ejecting crystals directly from their crystallization wells eliminates the laborious and time-consuming manual harvesting of fragile protein crystals. We here made it possible with a very simple modification of the MiTeGen In Situ-1 crystallization plate, that consists in a light sanding of their edge pedestal (Figure 1a). This is enough to make the plate acoustically compatible with the Echo 550 liquid handler (Labcyte Inc.) and would not be needed if the plates were designed with acoustically compatible plastic. An acoustic compatible plate enables multiple acoustic harvests of crystals from different wells, directly to the X-ray diffraction data collection media (micro-meshes), (Figure 1b). Each harvested aliquot can then be combined with distinct chemicals, making combinatorial crystallography an obvious application. Our results demonstrate that acoustic harvesting is not merely a viable and gentle crystal harvesting technique, but it also makes protein crystal harvesting remarkably more efficient.

In recent years, serial crystallography has emerged as promising method for structural studies of integral membrane proteins. The possibility of collecting data from very small crystals at room temperature, with reduced radiation damage, has opened new opportunities to the membrane protein structural biology community. In particular in the field of time-resolved studies. However, one of the technical bottlenecks of the method is the production of large amounts of tiny optimized crystals in mesophases. Here, we present a simple and fast method to prepare hundreds of microliters of high-density microcrystals in lipidic cubic phase (LCP) for serial crystallography including time resolved measurements. This approach not only eliminates the need for large quantities of expensive gas-tight syringes, but also may be used as a high-throughput tool when screening conditions for the growth of high density well-diffracting crystals.

We also demonstrate, with practical examples, that this new approach is of great advantage to fragment drug discovery since it facilitates in situ crystal soaking with minimal disturbance to the crystals in LCP.

Finally, the method is economical and easily implemented in any standard crystallisation laboratory.

Antibiotic resistance is growing to dangerously high levels and poses a serious threat to global public health. The emergence and spread of resistance mechanisms to all antibiotics introduced into the clinic jeopardize the effectiveness of current treatments. Traditionally, antibiotics have been designed to inhibit bacterial viability or impair their growth; these mechanisms induce a strong selection pressure for resistance development. To overcome this problem, an alternate approach is to disarm bacterial virulence without killing them, which potentially reduces selection pressure and delays the emergence of resistance.

In this project, we target the thiol-disulfide oxidoreductase enzyme DsbA which catalyzes disulfide bond formation in the periplasm of Gram-negative bacteria. DsbA facilitates folding of multiple virulent factors and acts as a major regulator of bacterial virulence. Bacteria lacking a functional DsbA display reduced virulence, increased sensitivity to antibiotics and diminished capacity to cause infection in many Gram-negative pathogens [1].

We carried out a fragment screening campaign against Escherichia coli DsbA and identified the first small molecule inhibitors that bind to the catalytic site of DsbA and inhibit DsbA activity in vitro and cell-based assays [2,3,4]. By exploiting an array of biophysical/biochemical tools (NMR, SPR, X-ray crystallography and in vitro assays), we aim to optimize these DsbA inhibitors from fragment hits to high-affinity leads. Herein we report our established drug discovery pipeline and current efforts in developing DsbA inhibitors. The goal of this work is to develop a new generation of antimicrobials with a novel mode of action that could be used alone or in combination with existing drugs to treat multi-drug resistant infections.

Crystallographic fragment screening (CFS) is an established method in academia and the pharmaceutical industry thanks to dedicated workflows established and optimized at several synchrotron sites. Apart from the hit identity, this technique also provides the structural 3D-information of the fragment hits on the protein surface and therefore fosters rational tool compound development and drug discovery.
At Helmholtz-Zentrum Berlin (HZB), a dedicated CFS-workflow is available that enables stream-lined experiments and data analysis [1]. The outcome of such a workflow depends to a large extend on the quality of the fragment library employed. Higher count and chemical diversity of the resulting fragment hits increases the chances for successful design of follow-up compounds. To this end, in collaborative effort with the drug design group at Marburg University, we designed libraries that are highly diverse in terms of their 3D‑pharmacophores and representative for the chemical space of fragments. The resulting F2X‑Universal Library of 1103 compounds and its representative sub-selection of 96 compounds - the F2X‑Entry Screen - are now the libraries of choice for CFS user campaigns at our facility due to their high performance [2]. Validation campaigns and several user campaigns were performed and showed hit rates of usually 15-25%, reaching 30% in exceptional cases. Another advantage of the libraries is their physical presentation as ready-to-use 96-well plates with dried-in compounds which also enables CFS for sensitive crystals without DMSO tolerance.
Apart from the exceptional library, a dedicated tool was developed at HZB to ease handling of large amounts of crystals - the EasyAccess Frame [3]. By supplying the users with such and other tools, as well as the dried-in libraries we can provide CFS campaigns as “fragment screening to go”, i.e., campaigns can be conducted inside our facility or in the user’s home laboratory. Furthermore, the robot assisted, and remotely controllable beamlines BL 14.1 and BL 14.2 are a key part of the HZB workflow and deliver high-quality diffraction data. Subsequently, processing of the data up to the identification of even weakly bound fragments is highly automated and provided via FragMAXapp, a user friendly, web-based tool developed in collaboration with the FragMAX facility at MAX IV [4]. Several CFS campaigns have been conducted successfully using the described workflow at HZB. As part of the EU-funded iNEXT Discovery project, application for access to the facility is straightforward and convenient for users from academia and industry.

[1] Wollenhaupt, J., Barthel, T., Lima, G.M.A., Metz, A., Wallacher, D., Jagudin, E., Huschmann, F.U., Hauß, T., Feiler, C.G., Gerlach, M., Hellmig, H., Förster, R., Steffien, M., Heine, A., Klebe, G., Mueller, U. & Weiss, M.S. (2021). J. Vis. Exp. 169, e62208
[2] Wollenhaupt, J., Metz, A., Barthel, T., Lima, G.M.A., Heine, A., Mueller, U., Klebe, G. & Weiss, M.S. (2020). Structure. 28, 694.
[3] Barthel, T., Huschmann, F.U., Wallacher, D., Feiler, C.G., Klebe, G., Weiss, M.S. & Wollenhaupt, J., (2021). J. Appl. Cryst. 54, 376.
[4] Lima, G.M.A., Jagudin, E., Talibov, V.O., Benz, L.S., Costantino, M., Barthel, T., Wollenhaupt, J., Weiss, M.S. & Mueller, U. (2021). Acta Cryst. D. accepted.

The hybrid inorganic–organic material [(CH₃)₂NH₂]₂NiCl₄ was reported to exhibit a remarkable thermochromism. [1] The color of this compound rapidly changes from deep red to deep blue upon heating at 383 K. Surprisingly, upon cooling to room temperature, the deep blue compound changes its color to dark, golden yellow. The so-produced yellow compound spontaneously transitions back to the starting deep red compound upon prolonged storage at ambient conditions. This color-change sequence can be cycled for a number of times without apparent degradation. Originally, it was believed that the color change originates from temperature-induced changes in the local geometry around the Ni⁺² cations in the structure. To shine light at these processes, we performed detailed studies using synchrotron X-ray powder diffraction, with diffraction data collected as a function of temperature. We discover that rather than undergoing thermochomic transitions, this compound is in fact a reacting system and the different color originate from different crystalline phases. The crystal structure and composition of these phases was solved and refined using the diffraction data. These structures were used to rationalize the color changes. This contribution emphasized the importance of powder X-ray diffraction, and crystallography in general, in the mechanistic studies of the stimuli-responsive crystalline compounds.

Molecules that change their colour, structure, and electronic properties in response to an external stimulus represent an emerging class of ‘smart’ material with potential applications in sensing, actuating and responsive technologies. The spin crossover (SCO) phenomenon leads to a redistribution of electrons within the d-orbitals of some transition metal complexes as a result of an external perturbation such as changes in temperature, pressure changes and light irradiation. The transition between high spin and low spin states involves a significant change in molecular volume and is often cooperative in crystalline materials, leading to dramatic changes in the optical, mechanical and magnetic properties.

We have demonstrated the use of mechanochemistry in the synthesis of SCO materials,¹ and have recently shown that they can be synthesised through contact of the reagents in the solid state without any applied mechanical force.² Recent work in our group has shown the significant promise of using supramolecular interactions to design new SCO materials with tunable thermally-responsive properties.³ This talk will focus on how various stimuli can affect the synthesis, structure and properties of these SCO materials in the solid state.

Rotors, motors and switches in the solid state find a favorable playground in porous materials, such as Metal Organic Frameworks (MOFs), thanks to their large free volume, which allows for fast dynamics. We fabricated MOFs with reorientable linkers and benchmark mobility also at very low temperature, to reduce the energy demand for motion-activation and light stimulus-response.

In particular, we have realized a fast molecular rotor in the solid state whose rotation speed approaches that of unhindered rotations in organic moieties even at very low temperatures (2 K). The rotors were hosted within the struts of a low-density porous crystalline MOF and energetically decoupled from their surroundings. A key point was the unusual crossed conformation adopted by the carboxylates around the pivotal bond on the rotor axle, generating geometrical frustration and very shallow wells along the circular trajectory. Continuos, unidirectional hyperfast rotation with an energy barrier of 6.2 cal/mol and a high frequency persistent for several turns is achieved (10 GHz below 2 K).[1]

Responsive porous switchable framework materials endowed with light-responsive overcrowded olefins, took advantage of both the quantitative photoisomerization in the solid state and the porosity of the framework to reversibly modulate the gas adsorption in response to light. [2]

Motors were inserted into metal-organic frameworks wherein two linkers with complementary absorption-emission properties were integrated into the same materials. Therefore, unidirectional motion was achieved by simple exposure to sun-light of the solid particles, which thus behave as autonomous nanodevices.[3]

MOF nanocrystals comprising high-Z linking nodes interacting with the ionizing radiation, arranged in an orderly fashion at a nanometric distance from diphenylanthracene ligand emitters showed ultrafast sensitization of the ligand fluorescence, thus supporting the development of new engineered scintillators.[4]

References

1. J. Perego, S. Bracco, M. Negroni, C. X. Bezuidenhout, G. Prando, P. Carretta, A. Comotti, P. Sozzani Nature Chem. 2020, 12, 845.

2. P. Sozzani, S. Bracco, S. J. Wezenberg, A. Comotti, B. L. Feringa et al. Nature Chem. 2020, 12, 595.

3. W. Danowski, F. Castiglioni, A. Comotti, B. L. Feringa et al. J. Am. Chem. Soc. 2020, 142, 9048.
4. J. Perego, F. Meinardi, S. Bracco, A. Comotti, A. Monguzzi et al. Nature Photonics 2021, doi 10.1038/s41566-021-00769-z.

Molecular crystals can be bent elastically by simultaneous expansion and contraction or plastically by delamination into slabs that glide along slip planes [1,2]. Here we describe a hitherto unreported mechanism of crystal bending in terephthalic acid crystal which undergoes pressure-induced phase transition upon bending where the two phases (form II and form I) coexist at ambient conditions. Scanning electron microscopy and microfocus XRD using synchrotron radiation provided direct evidence that upon bending, terephthalic acid crystals can undergo a mechanically induced phase transition without delamination and their overall crystal integrity is retained [3]. We report a distinctly different mechanism of plastic bending of molecular single crystals which have two phases and we provide the crystal structure of the bent section of such plastically bent crystal as direct evidence of the proposed mechanism. We also establish that this plastic deformation which effectively results in coexistence of two phases in the bent section of the crystal is the origin of unconventional properties such as shape-memory and self-restorative effects. Such plastically bent crystals act as bimorphs and their phase uniformity can be recovered thermally by taking the crystal over the phase transition temperature. This recovers the original straight shape and the crystal can be bent by a reverse thermal treatment, resulting in shape memory effects akin of those observed with some metal alloys and polymers. We anticipate that similar memory and restorative effects are common for other molecular crystals having metastable polymorphs.

[1] Ahmed, E., Karothu, D. P. & Naumov, P. (2018). Angew. Chem. Int. Ed. 57, 8837. [2] Naumov, P., Chizhik, S., Panda, M. K., Nath, N. K. & Boldyreva, E. (2015). Chem. Rev. 115, 12440. [3] Ahmed, E., Karothu, D. P., Warren, M. & Naumov. P (2019). Nat. Commun. 10, 3723.

Laser beam machining (LBM) of ceramics, polymers, or metals is usually performed using high-power femtosecond lasers (4–20 W). Using LBM, micro- or nano-sized patterns can be machined into surfaces of these materials to alter their properties for various applications. A drawback of such high-power techniques is the possibility of considerable chemical damage to the surface of the machined materials.

We now report the use of halogen bonding to generate new dye-based cocrystals with volatile cocrystal-forming molecules (coformers) that can be etched, cut, and punctured with micrometer-scale precision using low-powered laser beams (for example, between 0.5 and 20 mW).[1] This unique phenomenon, shown to be wavelength-tunable and power-dependent, can be utilized to machine molecular crystals by forming holes or cuts of controllable sizes. Using a microscope-guided low-power laser beam numerical control of this process can be achieved, enabling a variety of complex patterns to be inscribed onto the surface of molecular cocrystals. A mechanism is proposed with the volatile conformer acting as a leaving group, giving the ability to gently inscribe patterns using a low-power laser beam, without chemical decomposition of the cocrystals. This has not been previously reported in small molecule organic solids and appears to be a new emergent property achievable through crystal engineering by halogen bonding, opening a new type of materials to micrometer-scale shaping and machining applications.

[1] Borchers, T. H., Topić, F., Christopherson, J. -C., Bushuyev, O. S., Vainauskas, J., Titi, H. M., Friščić, T. & Barrett, C. J. (2021). ChemRxiv. https://doi.org/10.26434/chemrxiv.14398856.v1

Mechanical crystals are expected to be applicable for actuators and soft robots. Before the past decade, we have developed many mechanical crystals based on photoisomerization, and some based on phase transition and photothermal effect. In 2019, we have found a new kind of phase transitions, referred to as the photo-triggered phase transition. The photochromic crystal exhibiting a thermal, reversible single-crystal-to-single-crystal phase transition upon heating and cooling, transform to the identical phase upon light irradiation at temperatures lower than thermal phase transition temperature. A chiral salicylidnephenylethylamine [enol-(S)-1] crystal is known to undergo photoisomerization, and thermal phase transition. We have found that the enol-(S)-1 crystal exhibited the photo-triggered phase transition.

Upon heating, the enol-(S)-1 crystal in the α-phase (P2₁) transformed to the β-phase (P2₁2₁2₁) with the discontinuous β-angle change to 90° at 0 °C due to thermal phase transition from monoclinic to orthorhombic crystal system. Under UV light (365 nm) irradiation, the α-phase changed to the β-phase even at -30 °C. The mechanism was revealed that the photo-triggered phase transition is driven by the strain near the irradiated surface produced by the photoisomerization. A thick crystal in the α-phase deformed by the photo-triggered phase transition to the β-phase upon UV light irradiation; the surface temperature did not reach the thermal phase transition temperature. Furthermore, the thin plate-like crystal exhibited two-step bending motion by the photo-triggered phase transition and then the photoisomerization. Finally, by alternate irradiation of UV and visible light (488 nm) from the left, the plate-like crystal on the glass surface locomoted in the lower right direction. This finding leads to generalize the photo-triggered phase transition phenomenon and indicates that the photo-triggered phase transition enables to create various motions of crystals such as locomotion.