The major part of condensed matter in the Universe - deep inside planets and stars - exists under ultra-high pressures of several hundred gigapascals (GPa) and beyond. At such extreme conditions theoretical modelling predicts very unusual structures and chemical and physical properties of materials. Their synthesis and characterization at above 150 GPa have been hitherto hindered by the technical complexity of experiments involving samples’ heating and by a lack of relevant methods of the composition and structure investigations. Here on examples of simple elements, hydrides, oxides, carbonates, nitrides and silicates we will discuss single crystal X-ray diffraction experiments at static pressures from about 150 GPa to over 900 GPa in a laser-heated conventional and double-stage diamond anvil cells (ds-DAC).

Atomic model refinement and completion at low resolution (cryo-EM or crystallographic) is often a challenging task. This is mostly because the experimental data aren’t sufficiently detailed to describe using atomic models. To make refinement practical and ensure a refined model is geometrically meaningful additional a priori information about model geometry needs to be used. This information includes restraints on Ramachandran plot distributions or side chain rotameric states. However, using Ramachandran plot or rotameric states as refinement targets diminish the validating power of these tools. Therefore finding additional model validation criteria that are not used or difficult to use as refinement goals is desirable. Hydrogen bonds are one of most important non-covalent interactions that shape and maintain protein structure. These interactions can be characterized by specific geometry of hydrogen donor and acceptor atoms. Systematic analysis of these geometries performed for all quality-filtered high-resolution models of proteins from PDB shows they have distinct and conserved distribution that can be characterized by only two parameters. Here we demonstrate how these two parameters can serve as unique validation metrics and how they can pinpoint severe modeling problems that no other validation tools can detect. This tool is now a part of Phenix model validation suite; guidelines to its use and interpretation will be given.

Hybrid and integrative modeling methods that combine computational molecular mechanics simulations with experimental data are powerful in describing the structure and dynamics of large biomolecules. In particular, flexible fitting is a powerful technique to build the 3D structures of biomolecules from cryo-electron microscopy (cryo-EM) density maps. While flexible fitting methods work nicely with very high-resolution maps, there are limitations for medium resolution maps (~5-10 angstrom) in the case of complex conformational transitions. To overcome such issues, we proposed a refinement based on conformational ensemble, i.e., performing multiple fittings trials using various parameters. An automatic adjustment of the biasing force constants during the fitting process was introduced via a replica-exchange scheme to improve the success rate. From such multiple fittings, clustering analysis of the models obtained can be an effective approach to avoid over‐fitting. In addition, we have looked into the pixel size parameter as it can impact the resolution and accuracy of a cryo-EM map, and we proposed a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP score. We have demonstrated the efficacy of this protocol in retrieving appropriate pixel sizes via several examples.

Structural biology, the study of the 3D structures of biological entities on scales from small molecules to cells, has had an enormous impact on our understanding of biology and biological processes in health and disease. The results of these structural studies (mainly by MX, NMR and 3DEM) have been captured in the single global archive of atomistic models of biomacromolecules and their complexes, the PDB, operated by the wwPDB consortium. In addition, since 2002 the cryo-EM community has been depositing their maps and tomograms in EMDB.

A few years before the resolution revolution, wwPDB and EMDB jointly convened an EM Validation Task Force (VTF) which met in 2010 to discuss initial recommendations (published in 2012) regarding validation of cryo-EM data and models. In the following decade, the resolution revolution happened, EMDB grew from 1,000 to 15,000 entries, an archive for raw cryo-EM data was established (EMPIAR), community challenges related to EM validation were organised, and many labs began to develop new approaches to validating EM structures. This made it clear that a second EM VTF meeting was urgently needed. This meeting took place (in person!) in January 2020. During two days several dozen experts from all over the globe discussed cryo-EM data
management, deposition and validation.

A white paper summarising the discussions and recommendations of the second EM VTF meeting is currently in preparation. I will provide an overview of the major consensus recommendations emanating from the meeting and also address how wwPDB and EMDB are implementing these.

Cryo-EM is becoming an increasingly popular method of structure determination in structural biology. As the number of cryo-EM structures increases, it is important to maintain standards that measure the quality of those structures. The correctness of atomic models is very important because they often serve as targets for novel drugs or the knowledge base of such developments. Also, such standards are important to prevent the accumulation of errors of the structures in the databases. Thus, careful curation and validation of cryo-EM maps and derived atomic models are of utmost importance.

We have developed the EMDA Python package [1] that includes tools for cryo-EM map and model manipulation. In this presentation, the emphasis is given to those for validation. The majority of the current validation tools used in single-particle cryo-EM analyses are global metrics. They provide summaries of the global quality of maps or map-model fits. In order to reveal the local variation of the signal in maps and map-model fits, a new set of tools based on the local correlation have been developed. To calculate the local correlation, a spherical kernel is convolved with the map in image space to yield a correlation value at each voxel resulting in a three-dimensional (3D) correlation map. The variation of calculated correlation depends on the size of the kernel. The local correlation calculated using half maps captures the local variations in the signal, whereas the local correlation calculated between a map and a model indicates the quality of their fit. Map-model local correlation can be used to identify model regions outside the density or poorly fitted. Also, it can highlight unmodeled regions on the map. While the half map local correlation is useful to identify the presence/absence of the signal, its comparison with the map-model local correlation can be used to validate the map-model fit. In this presentation, we will demonstrate the use of local correlation through several examples. Also, EMDA includes several tools based on the maximum likelihood method. EMDA’s map-overlay and map magnification refinement are based on the maximisation of the joint probability distribution between two maps by a quasi-Newton method. We will demonstrate the use of map overlay and magnification refinement implemented in EMDA through examples.

To obtain more accurate atomic models from cryoEM and increase their impact on biomedical research, metrics are needed that carefully evaluate these constructed models. In this poster we present further developments on FSC-Q, a map-to-model quality issue recently introduced [1], with the capability to detect those areas of the model that are better supported by the experimental data (Figure1). The algorithm performs a careful analysis of the Signal-to-Noise Ratio in the half maps and in map generated from the proposed model through local resolution. It is intuitive and, yet, very precise, introducing quality information that we have quantitatively shown is new, in the sense that some of it was not captured in previous quality assessment metrics.

Electron cryo-microscopy (cryo-EM) is rapidly becoming a mainstream area of structural biology and medicine, enabling visualization and modelling of a wide variety of biologically important complexes. This recent explosion of new cryo-EM structures raises several important questions. How accurate are these maps and their model interpretations? What criteria are currently being used and are they good enough? This paper describes the outcomes of the 2019 Model Metrics Challenge sponsored by EMDataResource (https://challenges.emdataresource.org). The goals of this challenge were two-fold: (1) to evaluate the quality of models that can be produced using current modelling software, and (2) to assess the performance of metrics currently in use to evaluate cryo-EM models. In both instances the focus was on map targets selected the near-atomic resolution regime (1.8-3.1 Å), with an innovative twist: three of four maps formed a resolution series from the same specimen/imaging experiment.The results permit several specific recommendations to be made about validating near-atomic cryo-EM structures, both in the context of an individual laboratory experiment and for in the context of a structure data archive. We will also touch on preliminary results from our ongoing 2021 Ligand Model Challenge.

Q-scores are calculated locally for individual atoms in a model fitted or built into a cryo-EM map. They can be averaged over groups of atoms to represent resolvability of larger features such as residues in proteins, nucleotides in nucleic acids, and ligands. Plotting of residue or nucleotide Q-scores helps to identify which parts of a model are resolved in the map, and which parts may be unresolved or may need further refinement. A useful property of Q-scores is that for well-fitted models, they correlate strongly to the resolution of the map estimated by FSC; this answers the question ‘what is a good score’ for a map at a certain resolution. Several examples and related structural insights are shown with models and maps ranging from 2 to 5Å resolution. The connection between Q-scores and atomic B-factors is also explored. Finally, Q-scores are used to help detect and assess water and ion molecules in maps at 3Å and higher resolutions.

RNA viruses such as coronaviruses, flaviviruses, and henipaviruses represent major international health threats. Whilst these viruses replicate in the cytoplasm, they encode accessory proteins that target the host nuclear transport machinery to suppress innate immune pathways. Specifically, these virus proteins target the nuclear import receptor importin-a (IMPa) and inhibit host immune responses from entering the nucleus and triggering interferon (IFN) release. This immune evasion strategy is a critical component of virus pathogenicity, yet details of these interactions (including mechanism(s) of binding specificity with IMPa isoforms) remain unresolved. Here we describe the interfaces between these viral immune regulatory proteins and specific IMPA host receptors as targets for development of novel antivirals.

Prevalence of drug-resistant strains of causative agents of age old diseases pneumonia and tuberculosis (TB), has urged focus on exploring novel targets and development of new therapeutics with a fresh perspective in the battle against antibiotic resistance. Now-a-days bioactive compounds from natural origin are superseding the use of synthetic compounds due to structural and chemical diversity [1]. Our research illustrates the power of integrative structural biology in the discovery of inhibitors against two potential drug targets - (1) Serine acetyltransferase (also known as CysE), an enzyme of de novo cysteine biosynthetic pathway, and (2) Isocitrate lyases with role in both glyoxylate cycle and methylcitrate cycle

(1) CysE catalyzes the production of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine. The enzyme, essential for survival in a mouse model of TB infection [2], is absent in Homo sapiens. Therefore, this target is worth exploring for developing new antimicrobial compounds. The crystal structure of K. pneumoniae (Kpn) CysE was solved and used as a receptor for blind docking of natural compounds with documented antioxidant, antibacterial, respiratory stimulant, anti-inflammatory, and bronco-dilatory activities. L-Cys, a feedback inhibitor of CysE which binds at the active site was also docked as a positive control (Fig.1a). The best binders were tested for the inhibitory potential of CysE and quercetin was identified as the most potent inhibitor (Fig. 1b). MD simulations verified it as an allosteric inhibitor that binds at the trimer-trimer interface distal to the active and cofactor binding site.

(2) Isocitrate lyases (ICL1/ICL2) are essential for persistence of M. tuberculosis (Mtb) in its host [3] as they play an important role in metabolism of even and odd chain fatty acids via β-oxidation. Though high resolution crystal structures of Mtb ICL1 are available in PDB since 2000, and GlaxoSmithKline-TB Alliance launched high throughput screening of 900,000 compounds to identify ICL1 inhibitor, their efforts culminated in modest succes, in view of poor characterization of ICL2 structure-function relationship. We purified both Rv1915 and Rv1916 and characterized them possessing dual isocitrate and methylisocitrate lyase activities akin to ICL1[4]. In silico screening of natural compounds has yielded an inhibitor which is able to abolish both the activities in all Mtb ICLs.

[1]. Pereira D. M., Andrade C., Valentão P., & Andrade P. B. (2017). “Natural Products Targeting Clinically Relevant Enzymes, pp. 1–18. Wiley-VCH Verlag GmbH & Co. KGaA,

[2]. Sassetti C. M. & Rubin E. J. (2003).Proc. Natl. Acad. Sci. USA 100:12989-94

[3]. McKinney J. D., zu Bentrup K. H., Muñoz-Elías E. J., et al (2000). Nature 406:735–738.

[4]. Gould T. A., van de Langemheen H., Munoz-Elias E. J., et al (2006). Mol Microbiol 61:940–947.

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. This presentation will describe the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The Type II transporter has a tightly closed transmembrane channel due to ‘extracellular gating’ residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.

We know so little about how bacteria utilise surface virulence factors to colonise, infect, persist and cause disease in their hosts. The largest group of these virulence factors are the autotransporters, where although they employ a simple process for translocation to the bacterial surface, their functional passenger domains show a diverse range of pathogenic functions such as promoting adhesion, biofilm formation, invasion and tissue destruction. Despite extensive international efforts at the genotype-phenotype level that have confirmed the association of autotransporters with bacterial pathogenesis, less than 0.6 % of their structures have been determined with very little information on their molecular mechanisms of action.

With 10 new structures of autotransporter passenger domains over the past few years our group has been leading this area of research. Taking advantage of many autotransporter passenger domains being based upon large >500 residue β-solenoid structures, we have successfully employed Xenon derivatisation at the Australian Synchrotron to acquire anomalous signal for structure determination by single isomorphous replacement. More importantly, we have used our crystal structures to inform a comprehensive array of biophysical, biochemical and microbiological approaches to uncover the mode of action of the autotransporters and their roles in bacterial pathogenesis. Using this approach we were the first to determine the molecular mechanism of an autotransporter adhesin¹. We found that this Ag43 adhesin from Uropathogenic E. coli (UPEC) promoted bacterial biofilms through a self-association mechanism between neighbouring E. coli cell surfaces. This knowledge on biofilms is critical given their contribution to bacterial chronic infections and the development of antibiotic resistance.

Here we present the first crystal structure and mechanism of action of an autotransporter adhesin that binds to host tissue to facilitate bacterial colonisation². The crystal structure of UpaB from UPEC was found to display significant modifications to its β-helix that creates two different binding sites, allowing it to interact simultaneously with both host surface proteins and polysaccharides. As shown in live animal models, both sites co-operate to achieve bacterial colonisation. In contrast to Ag43 that forms self-associations that lead to biofilms, UpaB through directly binding host factors to facilitate colonisation creates a second mechanistic group of the autotransporter adhesins.

Returning to Ag43, we also investigate the conservation of its self-association mechanism with 3 new crystal structures of Ag43 homologues from widespread E. coli pathogens³. We show that adaptations to this mechanism of action alter the kinetics of bacterial aggregation and biofilm formation, presumably to suit the different E. coli pathogens to their specific infection sites. Even more importantly, we are using our molecular knowledge on autotransporters such as Ag43 to develop new classes of anti-bacterial inhibitors. To date we have developed and patented a successful inhibitor that targets Ag43 to prevent pathogenic E. coli biofilms⁴. Again using X-ray crystallography we have determined the structure of the first autotransporter adhesin-inhibitor complex to fully understand how this novel inhibitor interacts with Ag43 and blocks its funtion.

Figure 1A: Ag43 self-associates between E. coli surfaces to promote aggregation and biofilm formation. B. UpaB directly binds both host proteins and carbohydrates to promote UPEC colonisation.

[1] Heras B, Totsika M, Peters KM, Paxman JJ, Gee CL, Jarrott RJ, Perugini MA, Whitten AE and Schembri MA (2014). Proc Natl Acad Sci USA 111, 457-462.

[2] Paxman JJ, Lo A, Sullivan MJ, Panjikar S, Kuiper M, Whitten AE, Wang G, Luan CH, Moriel DG, Tan L, Peters KM, Gee C, Ulett GC, Schembri MA and Heras B. (2019). Nat. Commun. Apr 29;10(1);1967.

[3] Vo J, Martínez Ortiz GC, Totsika M, Lo A, Whitten AE, Hor L, Peters KM, Ageorges V, Caccia N, Desvaux M, Schembri M, Paxman JJ and Heras B (2021). ELife (under revision).
[4] Heras B, Paxman JJ, Schembri M and Lo A (2019) International Patent (PCT/AU2019/050893).

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The evolutionarily conserved Retromer complex (Vps35-Vps26-Vps29) is a master regulator responsible for endosomal membrane trafficking and signalling. It is known that mutations in Retromer can cause late-onset Parkinson’s disease, and can also be hijacked by viral and bacterial pathogens during cellular infection. Seeking tools to modulate Retromer function would provide new avenues in understanding its function and the associated diseases. Here we employed the random nonstandard peptides integrated discovery (RaPID) approach to identify a group of macrocyclic peptides capable of binding to Retromer with high affinity. Our crystal structures show that five of the macrocyclic peptides bind to human Vps29 via a di-peptide Pro-Leu sequence. Interestingly, these peptides structurally mimic known interacting proteins including TBC1D5, VARP, and the bacterial effector RidL, and potently inhibit their interaction with Retromer in vitro and in cells. In addition, we found that these Vps29-binding macrocyclic peptides also mimic the binding between thermophilic yeast Vps29 and the unstructured N-terminal domain of Vps5. Disruption of this previously uncharacterized interaction by macrocyclic peptides negatively affect yeast Retromer, Vps5 and Vps17 to form stable heteropentameric complex. By contrast, mutagenesis and cryoEM show that macrocyclic peptide RT-L4 binds Retromer at the Vps35 and Vps26 interface, and it can act as a molecular chaperone to stabilise the complex with minimal disruptive effects on Retromer’s ability to interact with its accessory proteins. Finally, using reversible cell permeabilization approach, we demonstrate that both the Retromer inhibiting and stabilizing macrocyclic peptides can specifically co-label Vps35-positive endosomal structures, and can be used as baits for purifying Retromer from cells and subsequent proteomic analyses. We believe these macrocyclic peptides can be used as a novel toolbox for the study of Retromer-mediated endosomal trafficking, and sheds light on developing novel therapeutic modifiers of Retromer function.

The class D serine β-lactamases comprise a superfamily of almost 900 enzymes capable of conferring high-level resistance to β-lactam antibiotics, predominantly the penicillins including oxacillin (Fig. 1) and cloxacillin, and some early generation cephalosporins. In recent years it has been discovered that some members of the class D β-lactamase superfamily have evolved the ability to deactivate carbapenems (Fig. 1), last resort β-lactam antibiotics generally held in reserve for highly drug resistant bacterial infections. These enzymes are collectively known as Carbapenem-Hydrolyzing Class D serine β-Lactamases or CHDLs [1,2]. Most alarmingly, a large number (>500) of these CHDLs have appeared in several Acinetobacter baumannii strains, leading the CDC to elevate this once nosocomial infection of little clinical importance into a major opportunistic pathogen, now deemed to be an urgent global threat [3] with mortality rates from infections by resistant strains often exceeding 50% [4].

The mechanism of β-lactam deactivation by the class D serine β-lactamases involves the covalent binding of the antibiotic to an active site serine to form an acyl-enzyme intermediate (acylation). This is followed by hydrolysis of the acyl bond (deacylation), catalysed by a water molecule activated by a carboxylated lysine residue [5]. It was initially thought that the carbapenems acted as potent inhibitors of the class D enzymes since formation of the covalent acyl-enzyme intermediate expelled all water molecules from the active site, and stereochemistry of the side group at carbon 6 of the β-lactam ring effectively blocked access into the pocket housing the catalytic lysine, thus preventing the deacylation step. Our recent structural studies on three CHDLs (OXA-23, OXA-48 and OXA-143) [4,6,7] have indicated that their carbapenem hydrolysing ability may be due to small-scale dynamics of two surface hydrophobic residues which form a hydrophobic lid over the internal pocket housing the catalytic lysine. Movement of one or both of these residues allow for the transient opening and closing of a channel (Fig. 2) through which water molecules from the milieu can enter the lysine pocket to facilitate the deacylation reaction. Although the hydrophobic residues responsible for the channel formation are present in all class D β-lactamases, sequence and structural differences nearby may be responsible for the evolution of carbapenemase activity in the CHDLs. Current and future work aimed at non-covalent inhibitor development in OXA-23, and improved covalent inhibitor design focused on blocking access to the catalytic lysine pocket in OXA-23 and OXA-48 will be presented.

[1] Queenan A.M. & Bush K. (2007). Clin. Microbiol. Rev. 20:440.

[2] Walther-Rasmussen J. & Hoiby N. (2006). J. Antimicrob. Chemother. 57:373.

[3] https://www.cdc.gov/drugresistance/biggest-threats.html

[4] Smith C.A., Antunes N.T., Stewart N.K., Toth M., Kumarasiri M., Chang M., Mobashery S. & Vakulenko S.B. (2013). Chem. Biol. 20:1107.

[5] Golemi D., Maveyraud L., Vakulenko S., Samama J.P. & Mobashery S. (2001). Proc. Natl. Acad. Sci. 98:14280.

[6] Toth M., Smith C.A., Antunes N.T., Stewart N.K., Maltz L. & Vakulenko S.B. (2017). Acta. Crystallogr. D73:692.

[7] Smith C.A., Stewart N.K., Toth M. & Vakulenko S.B. (2019). Antimicrob. Agents Chemother. 63:e01202-19.

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and to enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, over 1000 structures covering 18 of the SARS-CoV-2 viral proteins have been released in a span of a few months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination -and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force [1] has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others. We set up a website (www.insidecorona.net) and a database containing our evaluation and revised models; we met online every day, working on an automatic structure evaluation and revising individual structures. We also engaged in outreach activities, writing blog posts about the structural biology of SARS-CoV-2 aimed at both the scientific community and the general public, refining structures live on Twitch and offering a 3D printable virus model for schools. In the beginning, there were no tenured academics in the Coronavirus Structural Task Force; we were an ad hoc collaboration of 24 researchers across nine time zones, brought together by the desire to fight the pandemic. Still, we were able to rapidly establish a large network of COVID-19 related research, forge friendships and collaborations across national boundaries, spread knowledge about the structural biology of the virus and provide improved models for in-silico drug discovery projects.
[1] Croll, T., Diederichs, K., Fischer, F., Fyfe, C., Gao, Y., Horrell, S., Joseph, A., Kandler, L., Kippes, O., Kirsten, F., Müller, K., Nolte, K., Payne, A., Reeves, M.G., Richardson, J., Santoni, G., Stäb, S., Tronrud, D., Williams, C, Thorn, A*. (2021) Making the invisible enemy visible (2021) Nature Structural & Molecular Biology 28, 404–408 https://doi.org/10.1038/s41594-021-00593-7

This paper discusses, along with historical background, the principle and the proof-of-concept studies of crystalline sponge (CS) method, a new single-crystal X-ray diffraction (SCD) analysis that can analyze the structures of small molecules without sample crystallization. The method uses single crystalline porous coordination networks, called crystalline sponges, that can absorb small guest molecules into the pores. The absorbed guest molecules are ordered via molecular recognition in the pores and become observable by conventional SCD diffraction analysis. [[(ZnI2)3(tpt)2]•x(solvent)]n complex (tpt = tris(4-pyridyl)-1,3,5-triazine) was first proposed as a crystalline sponge and has been most generally used. The principle of the CS method can be described as “post-crystallization” of the absorbed guest, whose ordering is templated by the pre-latticed cavities. The method has been widely applied to synthetic chemistry as well as natural product studies, for which proof-of-concept examples will be shown here.

We have developed a novel approach called CrystalDirect that enables fully automated crystal mounting and cryo-coolingclosing the automation gap between crystallization and X-ray data collection. The CrystalDirect technology also allows the automated delivery of small molecules to crystals, giving access to large scale small molecule screening through X-ray crystallography. We have combined this approach with automated data collection at the ESRF and other synchrotrons to develop a fully automated, remote-controlled pipelines for macromolecular crystallography and an automated pipeline for large scale compound and fragment screening to support structure guided drug discovery programs. In order to facilitate high throughput data analysis, we have built a series of Application Program Interfaces (APIs) linking the Crystallization Information Management System (CRIMS) and the ISPyB system for automated synchrotron data collection with automated structure refinement and analysis, using software pipelines developed by Global Phasing. These pipelines effectively provide online access to crystallization and synchrotron diffraction and data analysis facilities and remove key bottlenecks in modern crystallography. They can contribute to the rapid progression of challenging projects in structural biology, to facilitate the access to protein crystallography for scientist of other disciplines and stimulate translation of basic research into biomedical applications. On the other hand, the large amounts of data generated pose new challenges, but also provide new opportunities to develop integrated systems for data acquisition, processing and analysis. The experience from the use of these pipelines as well as the new opportunities enabled by the integration of crystallization, X-ray data collection and analysis into continuous, fully automated workflows will be discussed.

Crystallographic fragment screening, which involves screening small-molecule libraries against crystals of a target protein, is an essential tool in modern drug discovery. The technique relies on the high-throughput generation of cryo-cooled, soaked crystals followed by fast and efficient data collection at a synchrotron beamline. Each campaign may generate hundreds or thousands of samples, and the most efficient strategy for acquiring data is to use unattended data collection followed by automatic data processing.

The macromolecular crystallography (MX) group at the Swiss Light Source operates a fast fragment and compound screening (FFCS) pipeline that uses Smart Digital User (SDU) software to collect data at the beamlines. This presentation will give an overview of SDU, describe how it has been implemented at the MX beamlines and present some recent case studies.

Structural Biology Research Center in the Photon Factory (PF), Japan, has five macromolecular crystallography (MX) beamlines at two synchrotron radiation rings, PF and PF-AR. The end stations of all the beamlines are equipped with sample exchange robots [1], high-precision diffractometers, and pixel array detectors, which are controlled by a common control software. This enables us to realize a fully automated and unattended data collection and a remote interactive data collection at all beamlines.

For a fully automated data collection, we developed a dedicated software SIROCC (Sophisticated Interface for Routine Operation with Crystal Centering). After mounting a new sample on the goniometer by the sample exchange robot, SIROCC recognizes a sample loop, and perform two raster scans over the loop regions. From a heatmap based on the number of diffraction spots below 4 Å resolution, SIROCC recognizes a shape and location of a protein crystal and places it to the X-ray beam position. By taking two snapshots, SIROCC evaluates the diffraction quality of the crystal, and if it exceeds a user’s defined threshold, SIROCC collects a complete diffraction data set. In a fully automated data collection beamtime, a beamline staff loads samples from users, and starts the automated data collection. Then all samples are mounted and diffraction measurements by SIROCC are performed in a fully automated manner. For a remote interactive data collection, NoMachine Workstation is installed on a workstation running the control software at each beamline. Furthermore, NoMachine Cloud Server, which federates the workstations at all beamlines, is installed on a gateway server which is accessible from outside the facility. At the beginning of a remote access beamtime, a beamline operating staff prepares the beamline, and give a permission for a user to access to the beamline through the gateway server. Then, the user connects through a NoMachine remote desktop software, allowing users to perform measurements remotely from outside the facility. All experimental information from the fully automated or remote data collection are recorded in the database system, PReMo [2]. PReMo also functions as a data processing and analysis pipeline, and users can obtain the experimental information and the result of data processing and analysis on the Web. Recently, a dedicated server for data download is opened, and users can download a diffraction data or data processing result to perform a further analysis with their own workstation immediately after the data collection.

In remote access or fully automated data collection, the user packs frozen samples in Uni-pucks and ships them to the PF using a dry sipper. The beamline staff receives the dry sipper and transfers to the beamline which the sample is assigned. The Uni-pucks are then taken out from the dry sipper and placed in the liquid nitrogen Dewar of the sample exchange robot. To prevent miss-match of the sample mount on the diffractometer in a fully automated measurement or/and remote experiment, the transportation of the dry-shipper and placing the Uni-puck must be performed without errors. Therefore, it is very important to establish a sample tracking system for the reliable operation of fully automated measurements and remote experiments. Recently, we developed a sample tracking system for those experiments. In this system, the user must attach QR code labels to items such as a dry shipper, hard disk drive and so on to be sent, and place a pin with a two-dimensional barcode into No. 16 of the Uni-puck. When the beamline staff takes any action on the shipped items, the QR code is always read, and the status of the item is updated. The status is stored in a database system and shared among beamline staff members, which helps to prevent miscommunication. The two-dimensional barcode on the pin on No. 16 is read by the sample exchange robot after the Uni-pucks are placed in the Dewar, and the robot recognizes automatically which Uni-puck is set in which position in the Dewar.

In 2020, due to the pandemic situation of COVID-19, approximately 75% of the beamtime in MX beamlines were used for remote experiments or/and fully automated measurements with users unattended.

[1] Hiraki, M., Yamada, Y., Chavas, L.M., Matsugaki, N., Igarashi, N. & Wakatsuki, S. (2013). J. Phys.: Conf. Ser. 425, 012014.[2] Yamada, Y., Matsugaki, N., Chavas, L.M., Hiraki, M., Igarashi, N. & Wakatsuki, S. (2013). J. Phys.: Conf. Ser. 425, 012017.

Keywords: Synchrotron, automation, remote access

This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED.

Watching biomolecules in motion on biologically relevant time scales has been a long-standing goal of structural biology. Current methodologies allowing for time-resolved crystallographic data collection are mostly through serial methods using microcrystals - which are technically challenging experiments with elaborate synchrotron beamline setups, consumption of large amounts of sample, and requiring contributions from many researchers. Here, we propose an alternative methodological setup in order to collect time-resolved data in the millisecond time regime (>5ms), suitable for measuring relatively large structural changes that may be rate-limiting in particular cases. Our approach has been to leverage rapid freeze-quenching by robotically plunging our crystals of choice through a substrate film prior to hyperquenching in liquid nitrogen. This method affords many quality of life improvements over current time-resolved methods, such as the potential for a single crystal use per time-point, divorcing the reaction initiation from data collection, and the ability to use the standard mail-in remote data collection available at all synchrotron sources. In order to show proof-of-concept, we used a well characterized metabolic enzyme phosphoenolpyruvate carboxykinase which converts oxaloacetic acid to phosphoenolpyruvate. Our initial experiments uncovered a previously hypothesized state believed to occur directly after phosphoryl transfer and prior to product release. We hope that this method, with its simplicity and ease of access, can allow many structural biology labs to begin time-resolved exploration of suitable systems to uncover further molecular details of enzymes of interest.

MXAimbot is a neural network based tool, designed to automate the task of centering samples for macro-molecular X-ray crystallography experiments before exposing the sample to the beam.

MXAimbot uses a convolutional neural network (CNN) trained on a few thousands images from an industrial vision camera pointed at the sample to predict suitable crystal centering for subsequent data collection.

The motivation for this project is that the machine vision automated sample positioning allows X-ray laboratories and synchrotron beamlines to offer a more efficient alternative for the manual centering, which is time consuming and difficult to automate with conventional image analysis, and for the X-ray mesh scan centering, which can introduce radiation damage to the crystal. MXAimbot can be used to improve results of standard LUCID loop centering for fully automated data collection in fragment-screening campaigns. No need for sample rotation should be an additional advantage.

A few original approaches and CNN architectures were tested by authors in [1,2]. They were using X-ray data from mesh scans and not relying on manual annotations. Finally for a current production a more simple method inspired by a DeepCentering approach [3] from SPring-8, has been adopted. The original training dataset was manually annotated with bounding-boxes around each crystal and the new CNN architecture is using the annotated data. MXAimbot can be used by other systems via a REST API. The next step for the project was including MXAimbot into MXCuBE3 - the common data acquisition framework at several European synchrotron facilities. This allows collection of anonymised datasets from the sample vision camera in the BioMAX beamline at the MAX IV synchrotron which can be further used for training and optimisation of CNNs and later be seamlessly included as an additional option in the MXCuBE3 data collection pipeline.

To the authors knowledge CNNs have been implemented for crystal centering at least at two synchrotron facilities including MAX IV. So far the CNN approach has shown outstanding results in automatically positioning crystals. Work is currently underway to test and statistically compare the model predictions to the manual centerings by real users with the goal of integrating MXAimbot into the FragMAX [4] - fragment screening facility at the MAX IV sychrotron.

[1] SCHURMANN, Jonathan; LINDHÉ, Isak. Crystal Centering Using Deep Learning. LU-CS-EX 2019-25, 2019.

[2] SCHURMANN, Jonathan; LINDHÉ, Isak et al. Crystal centering using deep learning in X-ray crystallography. Asilomar Conference on Signals, Systems, and Computers, 2019, 978-983. doi: 10.1109/IEEECONF44664.2019.9048793

[3] ITO, Sho; UENO, Go; YAMAMOTO, Masaki. DeepCentering: fully automated crystal centering using deep learning for macromolecular crystallography. Journal of synchrotron radiation, 2019, 26.4: 1361-1366. doi: 10.1107/S160057751900434X

[4] LIMA, Gustavo MA, et al. FragMAX: the fragment-screening platform at the MAX IV Laboratory. Acta Crystallographica Section D: Structural Biology, 2020, 76.8: 771-777. doi: 10.1107/S205979832000889X

In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge multiple data sets from one or more crystals, for example multiple small-wedge data collections on micro-crystals, in situ room temperature data collections, lipidic mesophase data collections or time-resolved crystallography. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, additional challenges are commonly encountered when merging multiple data sets. For novel structures, identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. The presence of non-isomorphous or poor-quality data sets may degrade the overall quality of the merged data set.

To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes as input the results of data sets individually integrated with DIALS [1] and performs symmetry analysis [2], scaling [3] and merging of multi-crystal data sets, as well as analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage [4] and preferred crystal orientation.

xia2.multiplex has been deployed as part of the autoprocessing pipeline at Diamond Light Source, including integration with downstream phasing pipelines such as DIMPLE [5] and Big EP [6].

Using data sets collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease, we demonstrate the use of xia2.multiplex within a wider autoprocessing framework to give rapid feedback during a multi-crystal experiment, and how the program can be used to further improve the quality of final merged data set.

[1] Winter, G., Waterman, D. G., Parkhurst, J. M., Brewster, A. S., Gildea, R. J., Gerstel, M., Fuentes-Montero, L., Vollmar, M., Michels-Clark, T., Young, I. D., Sauter, N. K. & Evans, G. (2018). Acta Crystallographica Section D.

[2] Gildea, R. J. & Winter, G. (2018). Acta Crystallographica Section D, 74(5), 405–410.

[3] Beilsten-Edmands, J., Winter, G., Gildea, R., Parkhurst, J., Waterman, D. & Evans, G. (2020). Acta Crystallographica Section D, 76(4), 385–399.

[4] Winter, G., Gildea, R. J., Paterson, N. G., Beale, J., Gerstel, M., Axford, D., Vollmar, M., McAuley, K. E., Owen, R. L., Flaig, R., Ashton, A. W. & Hall, D. R. (2019). Acta Crystallographica Section D, 75(3), 242–261.

[5] http://ccp4.github.io/dimple/

[6] Sikharulidze, I., Winter, G. & Hall, D. R. (2016). Acta Crystallographica Section A, 72(a1), s193.

Protein conformational dynamics play an important role in numerous biological processes. Markov State Models (MSMs) provide a powerful approach to study these dynamic processes by predicting long time scale dynamics based on many short molecular dynamics (MD) simulations. To improve the efficiency of MSMs, we recently developed quasi-MSM (qMSM) that encodes the non-Markovian dynamics in a generally time-dependent memory kernel. We successfully applied qMSMs to elucidate molecular mechanisms of DNA loading into a bacterial RNA polymerase complex via flexible loading gate (consisting of the clamp and β-lobe domain), a process occurs at millisecond. Using qMSMs, we showed that the opening of β-lobe is orders of magnitude faster than that of the clamp, which depends on the structure of the Switch 2 region. Strikingly, opening of the β-lobe is sufficient geometrically to accommodate DNA loading even when the clamp is partially closed. These two observations highlight β-lobe’s critical role allowing DNA loading during initiation. In my talk, I will also present our recent results in elucidating molecular mechanisms of 1′-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp).

COVID-19 pandemic is a great threat to the general and global public health and economy. The rapid development of new antiviral compounds and vaccines is needed to control the current pandemic as well as to prepare for the emergence of new variants. Among the proteins encoded by the SARS-CoV-2 genome, M^pro is one of the primary drug targets due to its essential role in maturation of the viral polyprotein. In this study, we describe a high-density acoustic droplet ejection (ADE) method for co-crystallization of M^pro-ligand complexes using only 40 nL M^pro solution. Also, we will briefly describe crystallographic data from crystals obtained using ADE and other methods as evidence that three clinically approved anti hepatitis C virus (HCV) drugs are capable of covalent binding to the M^pro Cys145 catalytic residue in the active site (Fig. 1). Activities of the National Virtual Biotechnology Laboratory (NVBL) for the design and development of new antiviral inhibitors for SARS-CoV-2 is briefly discussed.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness is responsible for more than half a million deaths in the United States alone. The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2’-OH of the first transcribed nucleotide (N₁ base) of the viral mRNA by covalently attaching a methyl group to it. This single RNA modification event converts the status of the mRNA cap from Cap-0 (^m7GpppA) to Cap-1(^m7GpppAm) and helps the virus evade immune surveillance in the host cell. Here, we report three high-resolution crystal structures of nsp16/nsp10 heterodimer representing substrate (Cap-0)-bound state, and pre- and post-release states of the RNA product (Cap-1). The binding of Cap-0 induces large conformational changes. This ‘induced fit’ model provides new mechanistic insights into the 2’-O methylation of the viral mRNA cap. We reveal the structural basis for the RNA specificity of nsp16/nsp10. We also discover an alternative ligand-binding site unique to SARS-CoV-2 [1]. We also observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate-binding region upon product release. These changes reset the enzyme for the next round of catalysis, and may be the structural basis of dissociation nsp10 from nsp16. The structures also identify a unique binding mode of a divalent metal ion in nsp16, which aligns the first two bases of the viral RNA in the catalytic pocket for efficient Cap-1 formation. Using LC/MS-based intact mass analysis, we show dramatic perturbations in Cap-1 formation by an emerging clinical variant of SARS-CoV-2, previous SARS-CoV outbreak strain, and their altered sensitivity to divalent metal ions [2]. Such reliance and preference for metals also suggests that an imbalance in cellular metal concentrations could differentially alter the RNA capping and thus, host innate immune response to infections by various CoVs. Altogether, our work provides a revised framework from which new therapeutic modalities may be designed for the treatment of COVID-19 and emerging coronavirus illnesses.

SARS-CoV-2 is a novel coronavirus and causative agent of the zoonotic disease Covid-19, which has been responsible for over 3 million deaths globally. Although the rapid development of several highly efficacious vaccines is proving effective in reducing the spread and severity of the disease, the development of novel, low cost and globally available anti-viral therapeutics remains an essential goal, both for this pandemic and for future outbreaks of related coronaviruses.

To identify starting points for such therapeutics, the XChem team at Diamond Light Source, in collaboration with various international colleagues, have performed large crystallographic fragment screens against 7 key SARS-CoV-2 proteins including the Main protease, the Nsp3 macrodomain and the helicase Nsp13 [1-3]. The expeditious collection and dissemination of data from these screens has been enabled by the well-established platform at Diamond and by the implementation of various new tools in the XChem pipeline.

This work has identified numerous starting points for the development of more potent inhibitors as exemplified by the ongoing work from the open science drug discovery project, the Covid Moonshot [4]. By merging fragment hits from the initial XChem screen and harnessing crowdsourced medicinal chemistry designs from the global community we have been able to rapidly develop potent inhibitors of the Main protease that exhibit promising antiviral activity.

[1] Douangamath, A., et al., Nature Communications, 11, 2020.

[2] Schuller, M., et al., Science Advances, 7, 2021.

[3] Newman, J., et al., BioRxiv, 2021.

[4] The COVID Moonshot Consortium, BioRxiv, 2021.

Human coronaviruses such as SARS-CoV, MERS and SARS-CoV-2 continue to emerge as significant threats to public health. Other human coronaviruses such as NL63, HKU1, 229E and OC43 continue to persist in the population but are significantly less deadly. Since the SARS-CoV epidemic emerged in 2003, we have worked to develop small-molecule inhibitors of coronavirus 3C-like protease (3CLpro, also known as main protease or Mpro) and the papain-like protease (PLP or PLpro). Initially, we focused on the proteases from SARS and then on NL63 and MERS. However, the differences in inhibitory potencies of our compounds and the taxonomic distance of the alpha and beta coronavirus genera taught us that approach of studying one virus at a time was too slow and provided to little molecular information to inhibit multiple coronaviruses. Moreover, it was not allowing us to predict how to inhibit emerging coronavirus pathogens. In the interest of pandemic preparedness, we are now taking what we call a taxonomically-driven approach to the structure-based design of coronavirus protease inhibitors. We targeted 12 different 3CLpros from the alpha-, beta- and gamma-coronavirus genera with a series of 50 compounds that we designed and synthesized using the Automated Synthesis and Purification platform at Eli Lilly. We identified inhibitor templates that potently inhibit the enzymes from the alpha and beta genera but not the gamma genus. To ascertain the structural basis of the selectivity, we utilized LS-CAT and LRL-CAT beamlines at the APS and performed a sparse-matrix sampling approach and determined multiple X-ray structures of 3CLpro from the different coronavirus genera in complex with different inhibitors. We identified precise structural regions that define inhibitor selectivity for different inhibitor scaffolds and we are now extending this approach to PLpro. We have been able to design and synthesize over 350 additional compounds against SARS-CoV-2 3CLpro. These compounds include potent non-covalent inhibitors, reversible-covalent and covalent inhibitors with low nanomolar to picomolar potency including inhibitors with broad-spectrum, i.e. pancoronavirus, activity against 12 different alpha, beta and gamma coronavirus.

This work was supported in part by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201700060C.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) is essential for the virus replication and covers multiple functions (1,2). In this context, PLpro is an interesting drug target to identify compounds that inhibit the activity and can further be optimized towards drugs to cure Covid-19 in the future. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, in terms of drug discovery investigations PLpro is thus an excellent drug target allowing a two-fold strategy, to identify compounds that inhibit viral replication and strengthen the immune response of the host in parallel. To establish a framework allowing an efficient and high throughput screening of compounds to identify inhibitors, we first expressed, purified and crystallized PLpro (Fig.1), determined and refined the native crystal structure to atomic resolution of 1.42 Å (Fig.2, pdb code: 7NFV).

Further, we initiated screening via co-crystallization utilizing a library of 2.500 selected natural compounds, obtained from ICCBS Karachi, and identified first potential inhibitors binding to a site that has been previously shown to bind to the ISG15 molecule, refined structures were deposited with pdb codes: 7OFS, 7OFT, 7OFU. Comparing the PLpro-ligand complex structures with the PLpro-ISG15 complex crystal structure (pdb code: 6XAA) clearly shows that several regions of the Ubiquitin fold domain move dynamically, showing functional flexibility to accommodate the ligands (Fig. 3). Corresponding structural data and details, as well as on-going structural efforts to identify new antiviral compounds to combat the coronavirus spread will be presented.

Hybrid structural methods to probe atomic features of the Type III Secretion Injectisome of Pathogenic Bacteria

Natalie C.J. Strynadka, Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T1Z3

Bacteria have evolved several sophisticated assemblies to transport proteins across their biological membranes, including those required specifically for pathogenicity. Recent advances in our understanding of the molecular details governing the molecular action of these protein secretion systems has benefited from an integrated toolbox of x-ray crystallography, NMR, mass spectroscopy, molecular modelling and increasingly and most dramatically, cryo electron microscopy. A syringe like nanoassembly, the Type III Secretion system injects multiple virulence “effector” proteins from the bacterial cytosol through to the infected host cell. These effectors manipulate host cell processes in varying ways to the benefit and subsequent pathogenicity of the bacteria. An essential element of disease in several of the most notorious Gram negative bacterial pathogens including the causative agents of food and water borne disease, hospital sepsis, cholera, typhoid fever, bubonic plague and sexually transmitted disease, a molecular understanding of the Type III Secretion systems being garnered from these structural studies provides the foundation for the development of new classes of antibacterials and vaccines to combat infection in the clinic and community. Highlights of recent advances in our structure/function analysis of the multi-membrane spanning Type III Secretion system “injectisome” will be presented emphasizing cryoEM focused refinements of the symmetry mismatched components of the core Type III Secretion System basal body complex spanning the inner through outer membranes of the prototypical Gram negative Salmonella typhimurium bacterial variant. These studies highlight a remarkable set of unexpected interactions including localized recruitment of protomers to allow symmetric coupling interactions between the inner and outer membrane components and a nanodisc like interaction of the inner membrane rings with the multicomponent export apparatus complex “gate”, T3SS proteins previously predicted to be membrane spanning in nature, but clearly sitting atop the membrane bilayer in the assembled structures.

References

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SctV cytosolic domain from the enteropathogenic E. coli T3SS injectisome. J
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The advances over the last decade in ultrabright X-ray sources have reinvigorated interest in time-resolved structural biology. Importantly, many of the time-scales of interest to structural biology are in fact accessible using serial crystallographic approaches at synchrotrons. Therefore, although the experiments themselves remain challenging, a number of new beamlines and endstations are coming online at synchrotron sources to serve this growing community. This has been complemented by increasing exchange between synchrotron and XFEL researchers, sharing methods, data processing tools and even supporting experiments that make use of both types of light source.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Electron density has been a great source of insight in the understanding of bonding and structure. Nonetheless, it lacks a fundamental characteristic: its connection to molecular and solid properties is barely predictive. This is so due to the lack of a direct (known) link between electron density and energetics.

Along this contribution we will try to fill this gap for several relevant cases in crystallography.

One way to approach this gap is to build energy models relying on topology. We have explored using a potential energy surface that includes chemical quantities explicitly, so that properties provided are directly related to the inherent organization of electrons within the regions provided by topological analysis [1]. Coupling this to conceptual DFT, the band gap of solids can be univocally defined [2]. Applied to zinc-blende solids as a model case, trends in band gap can be predicted in terms of bond properties (length, charge, crystalline structure- Figure 1).

Another field where energetic estimates from the electron density are missing is molecular crystals. This kind of approach would reveal extremely useful to predict the stability of different molecular crystal polymorphs or even cocrystals. In order to build this knowledge, we rely on the Non Covalent Interactions (NCI) index, which is able to identify the regions relevant to weak interactions from the electron density alone [3]. Simple approaches for well-known intermolecular energies datasets have allowed us to show that the energy can be predicted from these electron density regions using machine learning approaches in a fast and accurate manner [4]…next step is crystals!

[1] R. F. Borkman, R. G. Parr (1968) J. Chem. Phys. 48, 1116.

[2] J. Contreras-García, C. Cárdenas (2017) J Mol Mod 23, 271.

[3] E. R. Johnson, S. Keinan, P. Mori-Sanchez, J. Contreras-García, A. J. Cohen, and W. Yang, (2010) J. Am. Chem. Soc. 132 , 6498.

[4] F. Peccati, E. Desmedt, J. Contreras-García (2019) Comp. Theo. Chem., 1159, 23.

Intracellular protein crystallization occurs in many branches of life, yet the underlying cellular processes remain largely unknown. This is partly because of the scarcity of easily accessible, reproducible recombinant protein models that allow in-depth characterization of intracellular liquid-solid phase separation events. Such limitation prompts the need for identifying various classes of model proteins to examine the similarities, differences, or generalizability of such intracellular crystallization events. Furthermore, to exploit the potential values of cell-made protein crystals and the platforms to produce them, intracellular crystallization should first be understood using diverse classes of model proteins. After validating the individual crystallization events of cellular and viral proteins that readily crystallize in the ER, cytosol or nucleus, I demonstrate up to four independent crystallization events can take place concurrently in various combinations in different subcellular compartments of a single cell. For instance, by co-expressing NEU1 and human IgGs that undergo crystallization or liquid-liquid phase separation in the ER, I demonstrate two independent phase separation events can be simultaneously induced in the same continuous space of the ER lumen without mixing or interfering each other’s phase separation behaviors. Likewise, two concurrent crystallization events can take place in the cytosol or in the nucleus without mixing or interfering each other. Intracellular protein crystallization thus can happen in a crowded physiological cellular environment and does not require high protein purity. Furthermore, I report a simple method to increase the yield of intracellular protein crystals, in terms of crystal size and numbers, by treating the cells with a topoisomerase II inhibitor that blocks cell division without preventing cell size growth. This study not only presents accessible model tools for studying intriguing intracellular protein crystallization events, but also paves a way toward establishing methods and controlling the induction, quality, size, and yield of intracellular protein crystals for high-value proteins.

The recent advances of X-ray free electron lasers (XFEL) have enabled serial femtosecond crystallography (SFX) and structure determination for complex proteins such as membrane proteins in high resolution.^1-3Importantly, time-resolved (TR) studies have emerged allowing to assess their reaction dynamics. Initial demonstrations focused on light induced reactions; however, a large class of biological macromolecules acts by reaction with specific substrates requiring fast mixing approaches for TR-studies. Microfluidic tools in combination with common liquid injectors for protein crystals allow mixing times in the millisecond to second range, which is suitable to study the dynamics of enzymatic reactions with SFX at XFELs. A large drawback for TR-SFX with substrate-initiated reactions remains the large amount of protein and crystals needed to study the time evolution of a reaction. Every time point to be assessed requires a full data set which multiplies the amount of protein crystals needed by the number of time points to be studied. This may result in unsurmountable protein sample limitations requiring hundreds of mg of protein, which are not attainable for many proteins. Microfluidics allows to tackle this issue by reducing the required amount of protein sample. We propose to inject protein crystals with segmented flow approaches, which deliver crystals to the XFEL only when it pulses. We demonstrate how protein crystals in their mother liquor can be encapsulated in droplets surrounded by an immiscible oil and how these droplets can be intersected with an XFEL using common liquid jet injection methods. We demonstrated this approach reducing the amount of sample required to solve the room temperature structure of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) at the SPB/SFX instrument at the EuXFEL.⁴ Furthermore, we demonstrated the ability to electrically trigger the crystal laden droplet release in the microfluidic droplet generator, the interfacing of this approach with miniaturized optical droplet detection and an electronic feedback mechanism to tune the droplet release at a desired frequency matching the repetition rate of a particular XFEL instrument. This approach has been recently tested at the Macromolecular Femtosecond Crystallography instrument at the Linac Coherent Light Source, where the feedback mechanism was successfully implemented. Diffraction was recorded for lysozyme and the protein KDO8PS and the ability to tune the droplet release with a desired delay to the XFEL reference signal was also achieved. This is important to optimize the synchronization with the XFEL when implemented in particular chambers and various geometrical realizations of the droplet generator in relation to the XFEL interaction spot. In follow up experiments, we will assess the amount of sample that is required to obtain a full data set for KDO8PS and couple this strategy with microfluidic mixers, which have already been integrated into the 3D-printed droplet generators. With this approach, we predict that the amount of protein required to achieve a full data set can be reduced by nearly 90 %.

References:

(1) Spence, J. C. H.; Weierstall, U.; Chapman, H. N. Reports on Progress in Physics 2012, 75.

(2) Chapman, H. N.; Fromme, P.; Barty, A., et al. Nature 2011, 470, 73-U81.

(3) Martin-Garcia, J. M.; Conrad, C. E.; Coe, J., et al. Archives of Biochemistry and Biophysics 2016, 602, 32-47.

(4) Echelmeier, A.; Villarreal, J. C.; Kim, D., et al. Nat Comm 2020, 4511.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce proinflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional Xray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higherorder assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX¹.

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

.

Recombinant protein overproduction can lead to aggregation and aberrant artefacts due to the intrinsic specificities of proteins and the requirement of physiological factors (O₂ or light-sensitivity, partners, chaperones, cofactors and post-translational modifications requirements). The Wagner’s group (MPI Bremen) has developed a native shotgun approach baptized Crystallomics (Figure) to directly explore native protein complexes from anaerobic microorganisms that contain numerous exotic cofactors (e.g. iron-sulfur cluster).^1,2 After protein extraction, the soluble proteome is fractionated through successive chromatography types and the selective process of crystallisation is used as an ultimate purification step. Since this approach targets the most abundant proteins from the soluble fraction, a significant amount of protein crystals representative of the microorganism’s metabolic landscape have been obtained. Ab initio phasing was systematically used for the X-ray structure determination of these unidentified proteins. Crystals were sorted based on their colours. Sulfur-SAD^3,4 were performed on the transparent crystals by collecting high multiplicity and multi-orientations data at low energies on X06DA at the Swiss Light Source or on BL-1A at KEK. To reduce noise and improve the accuracy of the data quality some of the protein crystals were shaped with a deep-UV laser (to decrease X-ray absorption) and diffraction experiments were performed under helium environment with the recently developed PSI JUNGFRAU detector.⁵ For coloured crystals containing their native cofactors and heavy elements, X-ray fluorescent spectra were systematically measured. SAD were then performed at the edge of the atom of interest. Protein targets were then identified either by manual sequencing in the electron density maps or by fold similarity after reconstruction of a poly-alanine model.In complement to X-ray diffraction, in cristallo UV/vis absorption spectra were recorded by using the microspectrophotometer icOS⁶ at the ESRF to further investigate the nature of the state adopted by metal/absorbing centers. This synergistic approach proved that crystallisation not only separates proteins from each other but is also a powerful tool to isolate and characterized different protein states from a mixture.

[1] Vögeli B, Engilberge S, Girard E, Riobé F, Maury O, Erb TJ, Shima S, Wagner T. (2018) Proc. Natl. Acad. Sci. U S A. 27, 3380-3385.

[2] Engilberge S., Wagner T., Santoni G., Breyton C., Shima S., Franzetti B., Riobé F., Maury O., Girard E. (2019) J. Appl. Cryst.28, 722-731.

[3] Olieric V., Weinert T., Finke A. D., Anders C., Li D., Olieric N., Borca C.N., Steinmetz M.O., Caffrey M., Jinek M., Wang M. (2016) Acta Cryst. D72, 421-429.

[4] Basu S, Olieric V, Leonarski F, Matsugaki N, Kawano Y, Takashi T, Huang CY, Yamada Y, Vera L, Olieric N, Basquin J, Wojdyla JA, Bunk O, Diederichs K, Yamamoto M, Wang M. (2019) IUCrJ. 6, 373-386.

[5] Leonarski F, Redford S, Mozzanica A, Lopez-Cuenca C, Panepucci E, Nass K, Ozerov D, Vera L, Olieric V, Buntschu D, Schneider R, Tinti G, Froejdh E, Diederichs K, Bunk O, Schmitt B, Wang M. (2018) Nat. Methods.15, 799-804.

[6] von Stetten D, Giraud T, Carpentier P, Sever F, Terrien M, Dobias F, Juers DH, Flot D, Mueller-Dieckmann C, Leonard GA, de Sanctis D and Royant A. In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF. (2015) Acta Crystallogr. D, 71, 15-26.

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to get new high-quality structures. The crystallophore, Xo4, is a family of nucleating and phasing molecules based on lanthanide complexes. Tb-Xo4 was the first molecule of this family to be described [1].

Results obtained on more than fifteen proteins will be described and will show that Tb-Xo4 is an efficient tool to promote protein crystallization. Among these results, we will show that (i) Tb-Xo4 increases the number of crystallization conditions by promoting unique ones [1,2] (ii) the crystalline forms promoted by the crystallophore bypass crystal defects often encountered by crystallographers such as low-resolution diffracting samples or crystals with twinning [3] and (iii) the crystallization reproducibility is largely improved, a particular issue in structure-based drug design.

Contrary to the dogma that crystallization can only be promoted from pure protein sample, we have shown that crystals can be obtained from enriched fractions containing several proteins [3] leading to the structure determination of a protein complex [4]. Even more unexpected, the crystallophore is able to induce crystallization directly from the protein solution, as exemplified by the crystallization of hen egg white lysozyme in water [5].

Finally, we will also present preliminary results on several crystallophore variants showing complementarity with Tb-Xo4 thus enlarging the success in defining exploitable crystallization conditions.

Altogether, crystallophore is an efficient solution for protein crystallization and structure determination in the bio-crystallographer toolbox.

[1] Engilberge, S., Riobé, F., Di Pietro, S., Lassalle, L., Coquelle, N., Arnaud, C.-A., Pitrat, D., Mulatier, J.-C., Madern, D., Breyton, C., Maury, O. & Girard, E. (2017). Chem. Sci. 8, 5909–5917.[2] Jiang, T., Roux, A., Engilberge, S., Alsalman, Z., Di Pietro, S., Franzetti, B., Riobé, F., Maury, O. & Girard, E. (2020). Crystal Growth & Design. 20, 5322–5329.[3] Engilberge, S., Wagner, T., Santoni, G., Breyton, C., Shima, S., Franzetti, B., Riobé, F., Maury, O. & Girard, E. (2019). Journal of Applied Crystallography. 52, 722–731.[4] Vögeli, B., Engilberge, S., Girard, E., Riobé, F., Maury, O., Erb, T. J., Shima, S. & Wagner, T. (2018). Proceedings of the National Academy of Sciences. 115, 3380–3385.[5] de Wijn, R., Rollet, K., Engilberge, S., McEwen, A. G., Hennig, O., Betat, H., Mörl, M., Riobé, F., Maury, O., Girard, E., Bénas, P., Lorber, B. & Sauter, C. (2020). Crystals. 10, 65.

Keywords: Crystallisation; crystallophore; nucleating agents; structure determination; phasing.

Authors acknowledge financial supports from the Fondation Maison de la Chimie, Agence Nationale de la Recherche (ANR Ln23-13-BS07-0007-01) and Region AuRA for (program Xo4-2.0).

Crystallization in hydrogels is not a frequent practice in bio-crystallography, although the benefits are multiple: prevents convection and crystal sedimentation, acts as impurity filter, etc., and have been proven to be the cheapest means to produce protein crystals of high quality similar to those obtained under microgravity conditions [1-2]. Moreover, gel grown protein crystals are excellent candidates as seeds to produce crystals of bigger size for neutron diffraction or as media for crystals delivery in serial femtosecond crystallography [3].

Hydrogel should also be considered to exert control over the nucleation and growth processes. In this work we will present our most recent studies on the influence of agarose over the nucleation and growth of protein crystals. Crystal number and size was successfully tuned in a wide range of agarose concentration while keeping constant other conditions. Using five model proteins we demonstrate that the influence of gel content is independent of the protein nature, allowing the mathematical prediction of crystals flux and size with little experimental effort. The convection free environment obtained even at low agarose concentration [4] permits the obtention of high homogeneous micro-crystals slurries (Figure 1) that could be used for serial crystallography application [3] or for the mass production of enzyme crystals for industrial application [5]. Last, we will also show how it allows to explore the phase diagram under a kinetic regime that may facilitate the growth of different polymorphs.

Figure 1. Crystal size and number are fine-tuned using agarose as non-convective media during the crystallization process. (a) Proteinase-K crystals size as a function of agarose concentration and (b) as a function of precipitant concentration at fix agarose concentration of 0.1% (w/v). Scale bar is 100 µm.

[1] Gavira, J. A., Otálora, F., González-Ramírez, L. A., Melero, E., Driessche, A. E. S. v., & García-Ruíz, J. M. (2020). Crystals, 10, 68.[2] Lorber, B.; Sauter, C.; Théobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M.C.; Capelle, B.; Sanglier, S.; Potier, N.; Giegé, R. (2009). Prog. Biophys. Mol. Biol. 101, 13.[3] Artusio, F.; Castellví, A.; Sacristán, A.; Pisano, R.; Gavira, J.A. (2020). Cryst. Growth Des., 20, 5564.[4] Garcia-Ruiz, J.M.; Novella, M.; Moreno, R.; Gavira, J.A. (2001). J. Cryst. Growth, 232, 165.[5] Fernández-Penas, R.; Verdugo-Escamilla, C.; Martínez-Rodríguez, S.; Gavira, J.A. (2021). Cryst. Growth Des., 21, 1698.

Keywords: agarose hydrogel; protein crystal nucleation, serial crystallography

Supported by project BIO2016-74875-P (MINECO), Spain co-funded by the Fondo Europeo de Desarrollo Regional (FEDER funds), European Union.

Producing sufficient amounts or stable, properly folded protein is an essential prerequisite for protein crystallization, especially for challenging targets such as membrane proteins. However many proteins in their native form show limited stability and are refractory to expression in recombinant hosts. Over the last several years, ancestral sequence reconstruction has (ASR) emerged as a useful tool by which protein engineers and crystallographers can obtain highly robust forms of proteins of interest, which are often also expressed at relatively high levels in Escherichia coli. ASR involves inferring the ancestral state from an alignment of the sequences of extant forms of a given protein family, an evolutionary tree that represents their phylogeny, and an amino acid substitution model. We have developed a suite of software (GRASP: Graphical Representation of Ancestral Sequence Predictions) which enables the inference of ancestral protein sequences from alignments of up to ~ 10000 sequences using maximum likelihood, joint or marginal reconstruction methods. GRASP has been exemplified using several families of eukaryotic cytochrome P450 enzymes, membrane-bound, haemoprotein monooxygenases that have typically been challenging to express and crystallise. To date ASR has enabled expression of ancestral eukaryotic P450s at levels up to ~ 7 µM in E. coli cultures (~350 mg/L culture) leading to the successful crystallization of representative enzymes from several P450 subfamilies. While the resurrected ancestral proteins may not be identical to the extant proteins of principal interest, crystallization of ancestral homologues can provide insights into the structure of a protein family, including how to stabilize the protein fold. In addition, ancestors provide a robust and relevant template for structure function studies as well as protein engineering. When applied to previously uncharacterized sequences, ASR could enable the discovery of new folds and accelerate the functional and structural annotation of sequence-structure-function relationships in novel protein families.

Atomic crystal structure affects the electromagnetic and thermal properties of common matter. Similarly, the nanoscale structure controls the properties of higher length-scale metamaterials, for example nanoparticle superlattices and photonic crystals. We have investigated the self-assembly and characterization of binary solids that consist of crystalline arrays of 1) spherical viruses / other protein cages and 2) functional units [1]. The extremely well defined structure of protein cages (e.g. CCMV, TMV and ferritins) facilitates the construction of co-crystals with large domain sizes. The use of a second functional unit allows highly selective pre- or post-functionalization with different types of functional units, such as organic dyes [2,3], supramolecular hosts [4] and enzymes [5].

In the case of rod-like protein assemblies (e.g. tobacco mosaic virus), well-defined binary superlattice wires can be achieved [6]. The superlattice structures are explained by a cooperative assembly pathway that proceeds in a zipper-like manner after nucleation. Curiously, the formed superstructure shows right-handed helical twisting due to the right-handed structure of the virus. This leads to structure-dependent chiral plasmonic function of the material.

Our systematic approach identifies the key parameters for the assembly process (ionic strength, electrolyte valence, pH) and highlights the effect of the size and aspect ratio of the virus particles, which ultimately control the crystal structure and lattice constant. Protein-based mesoporous materials, nanoscale multicompartments and metamaterials are all applications that require such high degree of structural control.

[1] Kostiainen, M. A.; Hiekkataipale, P.; Laiho, A.; Lemieux, V.; Seitsonen, J.; Ruokolainen, J.; Ceci, P. (2013). Nat. Nanotech. 8, 52.

[2] Mikkilä, J., Anaya-Plaza, E., Liljeström, V., Caston, J. R., Torres, T.; De La Escosura, A. & Kostiainen, M. A. (2016). ACS Nano 10, 1565.

[3] Anaya-Plaza, E., Aljarilla, A., Beaune, G., Nonappa, Timonen, J. V. I., de la Escosura, A., Torres, T. & Kostiainen, M. A. (2019). Adv. Mat. 31, 1902582.

[4] Beyeh, N. K., Nonappa, Liljeström, V., Mikkilä, J., Korpi, A., Bochicchio, D., Pavan, G. M., Ikkala, O., Ras, R. H. A. & Kostiainen, M. A. (2018). ACS Nano 12, 8029.

[5] Liljeström, V., Mikkilä, J. & Kostiainen, M. A. (2014). Nature Commun. 5, 4445.

[6] Liljeström, V., Ora, A., Hassinen, J., Heilala, M., Hynninen, V., Joensuu, J., Nonappa, Rekola, H., Törmä, P., Ikkala, O., Ras, R. H. A. & Kostiainen, M. A. (2017). Nature Commun. 8, 671.

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications [1]. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase fold enzyme (T_m = 73.5°C; ΔT_m > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers [2]. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo [2]. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

References

[1] Markova K., Chmelova K., Marques S. M., Carpentier P., Bednar D., Damborsky J., Marek M. (2020). Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst. Chemical Science 11, 11162-11178.

[2] Markova K., Chmelova K., Marques S. M., Carpentier P., Bednar D., Damborsky J., Marek M. (2021). Computational protein stabilization can affect folding energy landscapes and lead to domain-swapped dimers. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.13634021.v1

L-asparaginases are used in the treatment of Acute Lymphoblastic Leukemia (ALL). However, the currently utilized bacterial-type enzymes cause several severe side effects. Therefore, there is an urgent need to develop a new generation of therapeutic L-asparaginases. Promising candidates can be found among plant-type L-asparaginases, which function as Ntn-hydrolases. Ntn‑hydrolases are produced as inactive precursors and develop enzymatic activity in an autoproteolytic maturation process. Unfortunetely, therapeutic use of wild-type (WT) plant-type enzymes is not possible due to their low substrate affinity (mM). Random mutagenesis is a protein engineering tool that can be used to enhance enzyme efficiency and study structure-function relationship. In this project, locally performed random mutagenesis was used to generate a library of mutants of plant-type L-asparaginase from E.coli (EcAIII). Several new variants of EcAIII were selected from the library and subjected to detailed structural (X-ray crystallography) and biophysical studies (nanoDSF, CD, activity/autoproteolytic tests).

The results of our studies revealed that autoproteolysis and enzymatic activity of EcAIII are unrelated events. Some variants of EcAIII retained the ability to autoprocess even in the absence of Arg207 (Fig. 1C), which is critical for catalysis and important for maintaining a H-bond network in the active site. Screening of thermal stability by nanoDSF showed that all analyzed unprocessed EcAIII variants had decreased T_m with respect to the WT enzyme. Thermal stability of variants cleaved into the mature aβ subunits varied, but some mutants with increased T_m were also found (Fig. 1A). Crystallization experiments proved that it was possible to obtain crystals of all variants cleaved into subunits. This was in stark contrast to the unprocessed mutants, which did not produce any crystals despite of extensive screening. This effect may be related to the presence of a highly disordered a-β linker in the uncleaved proteins. CD spectra showed that most of the unprocessed mutants are folded like the WT protein; however, some variants with significant changes in the CD signal were also found. The determined crystal structures (resolution 1.6-2.4 Å) showed that the active site of EcAIII is flexible enough to accept different amino acid substitutions; however, the type of substitution affects the H-bond pattern in the active site. Absence of Arg207 affects the overall conformation of the protein and leads to significant shifts of atomic positions in the entire enzyme molecule (Fig. 1B), as illustrated by the Cα rmsd value of 1.10 Å.

Work supported by National Science Centre (NCN, Poland) grants 2020/38/E/NZ1/00035 and 2019/03/X/NZ1/00584.

Pseudo symmetric, repeat proteins are favoured targets for computational protein design as they allow for the creation of larger domains with limited amino acids by exploiting their symmetric and repeating nature. One of the most common pseudo symmetric, repeat domains is the β-propellers fold. In addition, they fulfil many functions from sugar binding to enzymatic and protein-protein interaction mediation, thus increasing the potential applications of the designed proteins. Each propeller is built from 4-stranded anti-parallel β-sheets also known as a blade, repeated around a central axis. The number of blades differs from four to ten with seven and eight being the most common. The first successful computational protein design of a β-propeller was the 6-bladed Pizza protein¹. The RE3volutionary design method² makes use of ancestral sequence reconstruction and symmetry based template construction methods incorporated in Rosetta. Each blade of the pizza protein possess the same amino acid sequence. When two or three repeats of this sequence are expressed, they self-assemble into the 6-bladed domain.

The same design method was employed to design the eight or nine bladed Cake protein³. The protein consists repeating units of 42 amino acids, when eight repeats are expressed, the protein adopts the nine bladed fold. However, when nine repeats are expressed, the protein will adopt a nine bladed fold. This structural plasticity was unseen among β-propellers monomers. Its existence might explain the wide diversity of repeat numbers observed in β-propellers by allowing the change from even to odd numbers. Identical to the Pizza protein, smaller repeat fragments of Cake will self-assemble into either the eight-bladed protein or the nine-bladed protein. The structures of most these assemblies as well as the monomeric eight-and nine-bladed propellers were confirmed with X-ray diffraction experiments.

While the structural plasticity of the Cake protein is novel, we also wanted to create a protein that could only adopt the rare nine-bladed propeller fold. In order to achieve this a three-blade repeat (124 amino acids) was designed with a similar design strategy, with the idea the three-fold symmetry would prevent formation of eight bladed propellers. Two variants, Scone-E and Scone-R were created⁴. Crystallography revealed however that both designs adopted an eight-fold conformation. This failed design showcases that more research is needed to create a specific sequence for large β-propellers. In addition to this the Scone-E protein could only be crystallized upon addition of the polyoxometalate STA. This charged molecule interacts with multiple positively charged regions on the protein surface, neutralizing them. It can also bind multiple chains thus facilitating protein contacts, resulting in higher symmetric space groups.

Some of the designed proteins in this research behaved unexpectedly, thus illustrating the importance of accurate structure determination by X-ray diffraction to validate the designed proteins. In addition the design lessons on larger β-propellers could prove instrumental in the design of new functional proteins based on this common natural protein fold.

1. Computational design of a symmetrical β-propeller, Arnout R.D. Voet, Hiroki Noguchi, Christine Addy, David Simoncini, Daiki Terada, Satoru Unzai, Sam-Yong Park, Kam Y. J. Zhang, Jeremy R. H. Tame Proceedings of the National Academy of Sciences Oct 2014, 111 (42) 15102 15107; DOI: 10.1073/pnas.1412768111

2. Evolution-Inspired Computational Design of Symmetric Proteins, Arnout R. D. Voet, David Simoncini, Jeremy R. H. Tame, Kam Y. J. Zhang, Computational Protein Design, 2017, Volume 1529 ISBN : 978-1-4939-6635-6

3. Structural plasticity of a designer protein sheds light on β‐propeller protein evolution, Mylemans, B., Laier, I., Kamata, K., Akashi, S., Noguchi, H., Tame, J.R.H. and Voet, A.R.D. FEBS J, 2021, 288: 530-545. https://doi.org/10.1111/febs.15347

4. Crystal structures of Scone, pseudosymmetric folding of a symmetric designer protein, Bram Mylemans, Theo Killian, Laurens Vandebroek, Luc Van Meervelt, Jeremy R.H. Tame, Tatjana N. Parac-Vogt, Arnout R.D. Voet bioRxiv 2021.04.12.439409; doi:https://doi.org/10.1101/2021.04.12.439409

Fluorescent proteins (FPs) play an indispensable role in advanced imaging techniques. Such proteins are considered as “smart labels” allowing scientists to overcome the diffraction barrier of conventional light microscopy to visualize subcellular events. Since the first discovery of GFPs in the 1960s [1], numerous studies have been conducted to design new fluorophores not only covering the whole visible light from cyan to far-red region but also displaying improved photochemical performances. Furthermore, the FP technology gains remarkable achievements by developing successfully special classes of FPs which exhibit photo-transformable properties including photoactivation (PA), irreversible photo-conversion (PC) and reversible photo-switching (RS) [2]. Currently, irreversible photoconvertible and reversible photo-switchable FPs attract wide interest of scientists due to their potential of converting from an emissive state to another emissive state or switching between a fluorescent on and a non-fluorescent off state, respectively. The combination of reversibly switchable behavior and spectrally different emission has enabled application of multicolored super-resolution microscopy techniques in live-cell imaging. However, various drawbacks of currently used reversibly switchable red FPs (rsRFPs) have limited their application greatly and made them still being the least used in GFP-like proteins family. Moreover, the structure-function relationship and the mechanism controlling photo-switching behavior of rsRFPs have not been understood completely. Therefore, structural studies are essential to provide valuable information for the rational design of improved rsRFPs which fit better to experimental requirements.

The rsCherry protein was the first reported reversibly switchable red FP which was developed from mCherry – a good label in imaging techniques [3]. However, due to the non-optimal properties of rsCherry such as limited brightness, poor photostability and low contrast between on and off states [4]its application in super-resolution microscopy was not very widespread. Our current study has shown that rsCherry lost its absorption at 572 nm as well as fluorescence when it aged, despite being well protected from light, making studying its molecular structure and photo-mechanisms challenging. We were able to identify that the time-dependent bleaching in rsCherry is related to chromophore modifications and proposed that oxygen, a critical external reagent in the maturation process of FPs, is involved in unexpected chemical reactions of the chromophore. Spectroscopic data, native MS results and mutagenesis analysis, and especially structural studies of rsCherry crystallized in strictly anaerobic conditions strongly confirm our hypothesis that oxygen diminishes the rsCherry fluorescence through modifying its chromophore. These findings can help to develop improved red fluorescent proteins suitable for specific advanced imaging techniques.

Each of us has the potential to make trillions and more different antibodies and antigen receptors even though our genome contains only 3.2 billion base pairs! The antibody diversity is created by stochastic recombination of gene segments (V, D and J) that encode antibody heavy and light chains, and the process is known as V(D)J recombination. To initiate V(D)J recombination, the RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences (RSSs), the 12- and 23-RSS (12/23-RSS). DNA double strand cleavage is achieved in two consecutive steps, hydrolysis and strand transfer resulting in a DNA hairpin, in a single active site. Using X-ray crystallography and cryoEM, we have determined how two RSS DNAs are paired, nicked and completely cleaved at atomic resolution [1-3]. Both the protein and DNA undergo large conformational changes, and the active site of RAG1 re-arranges for DNA nicking and hairpin formation. Although RAG belongs to the RNH-type transposase family, RAG-catalyzed transposition is inhibited in developing lymphocytes. We show that by efficiently catalyzing the disintegration reaction that reverses the strand transfer, RAG avoids DNA transposition and consequent genome instability. The structures also rationalize many RAG mutations that cause immune deficiency in humans.

In each human cell, only a subset of the genes is transcribed into RNA and translated into proteins, because gene expression is tightly regulated. The regulation of gene expression is a complex process involving several hierarchical stages, beginning in the nucleus with the control of transcription initiation by transcription factors. After maturation, mRNAs are translocated into the cytosol, where their stability, translation and, ultimately, degradation is under the control of RNA-binding proteins. The central event at all stages of gene expression regulation involves the recognition of DNA or RNA sequence motifs by transcription factors or RNA-binding proteins. Structural studies in my laboratory have highlighted crucial aspects of transcriptional and translational regulation. Transcriptional target gene recognition in bacteria (1) and eukaryotes (2,3) proceeds according to common principles, but differs in crucial aspects. Half-life and translation of mRNAs are controlled by proteins often binding to the 3’-untranslated regions of mRNAs. RNA-binding proteins studied in my laboratory promote mRNA degradation by recruiting nucleolytic degradation complexes (4,5) or through their intrinsic ribonuclease activity (6).

References

1. Khare, D. et al. (2004) Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. Nat. Struct. Mol. Biol. 11, 656-663
2. Schuetz, A. et al. (2011) The structure of the Klf4 DNA-binding domain links to self-renewal and macrophage differentiation. Cell. Mol. Life Sci. 68, 3121-3131
3. Ming, Q. et al. (2018) Structural basis of gene regulation by the Grainyhead/CP2 transcription factor family. Nucleic Acids Res. 46, 2082-2095
4. Mayr, F. et al. (2012) The Lin28 cold-shock domain remodels pre-let-7 microRNA. Nucleic Acids Res. 40, 7492-7506
5. Schuetz, A. et al. (2014) Roquin binding to target mRNAs involves a winged helix-turn-helix motif. Nat. Commun. 5:5701
6. Garg, A. et al. (2021) PIN and CCCH Zn-finger domains coordinate RNA targeting in ZC3H1 family endoribonucleases. Nucleic Acids Res. 49, 5369-5381

The fine details of the electron density distribution, typically far beyond the possibilities of standard X-ray diffraction analysis, can be approached when (ultra)high-resolution diffraction data are available, to allow abandoning the standard model of independent, spherically symmetrical atoms. This more complicated, and much more demanding (both experimentally and computationally) method allows, for instance, to analyze the redistribution of electron density into bonds, intermolecular interactions, etc. Moreover, the Atoms-In-Molecules approach, which is based on the analysis of topological features of high-quality electron density distribution, may offer an insight into the hierarchy of interactions, energetic features, etc. Even though such an approach is well-developed for small molecules, its application in macromolecular crystallography is still under development. Ultrahigh resolution in this case means resolution of at least 0.7 – 0.65 Å. Such data are extremely rare for macromolecular crystals. In addition, modelling problems, disorder, high solvent content, etc., severely limit the number of successful studies of experimental electron density distribution in macromolecules, which so far have been reported for proteins only (e.g. crambin [1], aldose reductase [2] and the high-potential iron–sulfur protein [3]).

For the present study, ultrahigh-resolution diffraction data (0.55 A) were collected for a Z-DNA hexamer with the sequence d(CGCGCG)₂. The results of the high-quality standard refinement [4] suggested that bonding and other features are visible in the difference electron density map (Fig). The quality of these data indeed allows the application of the more sophisticated multipolar model.

Fig. 1. The Watson–Crick base pair C3×G10 with the corresponding Fo map (blue, 3s) and difference Fo-Fc map (green, 2s) calculated without the contribution of H atoms.

The multipolar model has been successfully constructed and the drop in the R factor (and R free) seems to show that real experimental features have been included in this model. The topology of the electron density distribution has been analyzed and the intra- and intermolecular interactions characterized. A number of problems related to the multipolar approach, together with some expected and some unexpected results (e.g. unusual disorder of one of the C bases) will be presented. We will also consider the practical question concerning this kind of research: is the additional burden and investment of effort justified by the results?

[1] C. Jelsch, M. M. Teeter, V. Lamzin, V. Pichon-Pesme, R. H. Blessing, C. Lecomte. PNAS 97, 3171-3176 (2000).

[2] B. Guillot, C. Jelsch, A. Podjarny, C. Lecomte. Acta Cryst. D64, 567-588 (2008).

[3] Y. Hirano Y, K. Takeda, K. Miki. Nature 534, 281–284 (2016).

[4]K. Brzezinski, A. Brzuszkiewicz, M. Dauter, M. Kubicki, M. Jaskolski, Z. Dauter. Nucleic Acids Res. 39, 6238-6248 (2011).

One of the kinds of information gained from high resolution (sub-atomic) structures is the observation that electron density parameters are transferable between atoms having similar chemical topology. This stimulated creation of databases of multipolar pseudoatoms (Invariom [1], ELMAM2 [2], MATTS – successor of UBDB [3], etc.) and their applications in (a) structure refinements on standard (atomic) resolution data for small-molecule crystals, and (b) electrostatic properties and non-covalent bonding characterisations for macromolecules.

Transferable Aspherical Atom Model (TAAM) of scattering built from a pseudoatom database proved to be advantageous in refining the structure on X-ray diffraction (XRD) data compared to the Independent Atom model (IAM) [4], leading to better fit of the model to the data and improved localization of hydrogen atoms. We have recently showed [5] that also for small-molecule 3D electron diffraction (3D ED) data, a better model-to-data fit and more accurate structures should be expected from TAAM.

To improve its usability, we further extended the MATTS bank to cover 98% of atoms found in all the structures deposited in the Cambridge Structural Database [6] composed of chemical elements like C, H, N, O, P, S, F, Cl and/or Br. It is planned that the remaining 1% will be covered by the more general atom types resulting from multidimensional cluster analysis.

Some benefits of TAAM over IAM refinements were also reported for macromolecular XRD data of 0.9 Å resolution and better [7]. As most macromolecular crystals diffract to lower resolutions, we recently moved our investigation towards near-atomic resolutions. We quantified the differences between the macromolecular electron density Fourier maps obtained with TAAM and IAM, calculated with a resolution of 1.8 Å. We did the same for electrostatic potential maps, a key property in the context of 3D ED.

TAAM refinements affect not only the positions of the atoms, but also the atomic displacement parameters (ADPs) [8]. ADPs appears to be less resolution dependent with TAAM than with IAM. With IAM, ADPs increased for XRD and decreased for 3D ED by about 30%, when the resolution was reduced from 0.6 Å to 0.8 Å [5]. From modified Wilson plots we recently predicted, and then verified by TAAM refinements on macromolecular XRD or 3D ED data, what will happen with ADPs (B-factors) with a further resolution worsening, up to 1.8 Å.

All the above helps to understand if there will be any benefits of TAAM refinements on lower than atomic resolutions.

[1] Dittrich, B., Hübschle, C. B., Pröpper, K., Dietrich, F., Stolper, T. & Holstein, J. (2013). Acta Crystallogr. B 69, 91. [2] Domagała, S., Fournier, B., Liebschner, D., Guillot, B. & Jelsch, C. (2012). Acta Crystallogr. A 68, 337. [3] Kumar, P., Gruza, B., Bojarowski, S. A. & Dominiak, P. M. (2019). Acta Crystallogr. A 75, 398. [4] Jha, K. K., Gruza, B., Kumar, P., Chodkiewicz, M. L. & Dominiak, P. M. (2020). Acta Crystallogr. B 76, 296. [5] Gruza, B., Chodkiewicz, M., Krzeszczakowska, J. & Dominiak, P. M. (2020). Acta Crystallogr. A 76, 92. [6] Groom, C. R., Bruno, I. J., Lightfoot, M. P. & Ward, S. C. (2016). Acta Crystallogr. B 72, 171. [7]. Malinska, M. & Dauter, Z. (2016). Acta Crystallogr. D 72, 770. [8]. Sanjuan-Szklarz, F. W., Woińska, M., Domagała, S., Dominiak, P. M., Grabowsky, S., Jayatilaka, D., Gutmann, M., Woźniak, K. (2020). IUCrJ, 7, 920.

Keywords: quantum crystallography; structure refinement; X-ray diffraction, electron diffraction; 3D ED, microED, aspherical scattering factors; multipolar model; TAAM; MATTS; UBDB; ELMAM2

Support of this work by the National Centre of Science (Poland) through grant OPUS No.UMO-2017/27/B/ST4/02721 and PL-Grid through grant plgubdb2020 is acknowledged.

Lipases (E.C. 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or ‘interfacial activation’, with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica Lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported leading to the conclusion that its behaviour was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution [1]. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. Once positioned within the catalytic triad, substrates are then hydrolysed, and products released. However, the intermediate steps of substrate transfer from the lipidic-aqueous phase to the enzyme surface and then down to the catalytic site are still unclear. By inhibiting CALB with ethyl phosphonate and incubating with glyceryl tributyrate (2,3-di(butanoyloxy)propyl butanoate), the crystal structure of the lipid-enzyme complex, at 1.55 Å resolution, shows the tributyrin in the limbus region of active site [2]. The substrate is found above the catalytic Ser, with the glycerol backbone readily pre-aligned for further processing by key interactions via an extended water network with α-helix10 and α-helix5. These findings explain the lack of ‘interfacial activation’ of CALB and offer new elements to elucidate the mechanism of substrate recognition, transfer and catalysis of Candida antarctica Lipase B (CALB) and lipases in general.

[1] Stauch, B., Fisher, S. J., Cianci, M. (2015). Journal of Lipid Research, 56, 2348-2358.

[2] Silvestrini, L. & Cianci, M. (2020). International Journal of Biological Macromolecules, 158, 358-363.

Tryparedoxins are critical regulators of the redox metabolism in parasitic protozoa such as trypanosomones and leishmania, which cause the neglected tropical diseases sleeping sickness and leishmaniosis, respectively. Although tryparedoxins belong to the thioredoxin superfamily, they differ in their substrate specificity for the low molecular weight redox carrier, utilizing trypanothione, a spermidine-linked di-glutathione instead of glutathione. The unique nature of the redox carrier opens avenues for the targeted interference in the protozoan redox metabolism which hold potential for future therapeutic intervention.

We were able to obtain crystals of a tryparedoxin which have the potential to diffract to ultra-high resolution. Crystals of the oxidized protein diffract to well below 1 Å with our current highest resolution data set extending to 0.75 Å/0.85 Å resolution in the best/worst direction. Preliminary refinement indicated a mixture of oxidized and reduced states in the Cys-Pro-Pro-Cys active site with photoreduction of the disulfide bond being the reason for the appearance of the reduced state. Subsequent efforts resulted in crystal structures of the oxidized and reduced states, which at present extend to a more limited resolution in the 1 to 1.1 Å range. An analysis of the two redox states defines the redox linked conformational changes in the tryparedoxin family.

The maps of electrostatic potential from cryo-electron microscopy and micro-electron diffraction are now being obtained at atomic resolution. This extends the possibility of investigating the electrostatic potential beyond determining the non-hydrogen atom positions, taking into account also the negative regions of the maps. However, accurate tools to calculate this potential for macromolecules, without reaching to the expensive quantum calculations, are lacking. Simple point charges or spherical models do not provide enough accuracy. Here, we apply the multipolar electron scattering factors and investigate the theoretically-obtained potential maps.

The multipolar electron scattering factors are derived from the aspherical atom types from Multipolar Atom Types from Theory and Statistical clustering (MATTS) databank (successor of UBDB2018 [1]). MATTS has been created since electron densities of atom types are transferable between different molecules in similar chemical environment. These atom types can be used to recreate the electron density distribution of macromolecules via structure factors [2] and to calculate the accurate electrostatic potential maps for small molecules [3]. MATTS reproduces the molecular electrostatic potential of molecules within their entire volume better than the simple point charge models used in molecular mechanics or neutral spherical models used in electron crystallography. In this study, we calculate electrostatic potential maps for several chosen macromolecules using aspherical atom databank and compare them with experimental maps from cryo-electron microscopy and micro-electron diffraction at high resolution. Calculations at different resolutions reveal at which spatial frequencies different elements become discernible. We also consider the influence of atomic displacement parameters on the theoretical maps as their physical meaning in cryo-electron microscopy is not as well established as in X-ray crystallography.

This study could potentially pave the way for distinguishing between different ions/water molecules in the active sites of macromolecules in high resolution structures, which is of interest for drug design purposes. It could also facilitate the interpretation of the less-resolved regions of the maps and also advise in simple yet questionable issue of resolution definition in cryo-electron microscopy.

The authors acknowledge NCN UMO-2017/27/B/ST4/02721 grant.

References:

[1] Kumar et al. (2019). Acta Cryst. A75, 398-408

[2] Chodkiewicz et al. (2018). J. Appl. Cryst. 51, 193-199

[3] Gruza et al. (2019), Acta Cryst. A76, 92-109

Monovalent inorganic gallium compounds are very rare, whereas this is not the case for indium compounds. As the chemistry of Ga(I) can be assumed to be dominated by its lone pair that favors unsymmetrical environments, the influence of the lone pair of In(I) is usually not very pronounced. Thus, there are very few isostructural Ga and In compounds. Starting from the elements, we have now obtained the new telluridogallates(I) REGaTe₂ and related compounds REInTe₂ (RE = La – Nd). Although their orthorhombic unit-cell dimensions are similar, the structures combine modular entities in different ways that enable more or less space for lone pairs. In the case of In-containing compounds, data were collected using microfocused synchrotron radiation from microcrystals that were selected and pre-characterized by electron microscopy

The compounds REGaTe₂ crystallize in the non-centrosymmetric space group Pmc2₁, their lattice parameters reflect the lanthanide contraction. In contrast to telluridogallates(III) such as CuGaTe₂ [1] and AgGaTe₂ [2] that contain [GaTe₄] tetrahedral, its characteristical structural feature is a chain of GaTe₃ pyramids sharing two Te atoms with neighboring pyramids. This would be typical for a telluridogallate(I), however, chemical bonding and charge distribution are not trivial. Bond valence sums confirm the electron-precise description according to RE^IIIGa^ITe^-II₂, and the coordination of gallium(I) is pyramidal as expected for a lone-pair atom. Bader charges for LaGaTe₂ (La +1.5, Ga +0.5, Te ‑0.8 – ‑0.9) suggest only partial electron transfer. Thus, REGaTe₂ are rare examples of compounds with exclusively monovalent Ga atoms, probably the first one without organic residues. In NdGaTe₂, a short Nd-Ga distance of 3.13 Å is a possible indication of an interaction of the lone pair of Ga(I) with the Nd atoms. This is stronger than, for example, in NdGaSb₂ (Nd-Ga distance 3.35 Å),[3] which has a different structure and bonding situation. Similar Nd-Ga distances are observed in intermetallic phases such as NdGa [4] or NdGaRh [5], so that a description as an oxidized intermetallic phase may also be considered. A formal consideration in the framework of the Zintl concept would assume a mixed chain-like [Ga^(‑2)Te⁽⁰⁾Te^(-1)]^3- polyanion; note that in these formal “charges” are note expected correspond to oxidation states. It is consistent with all descriptions that the distances of 2.67 Å to the terminal Te atom are shorter than those to Te atoms bridging along the chain (2.95 Å). Interactions between the polyanionic chains appear negligible. The Nd atoms are located in single-capped trigonal prisms of Te atoms, with Ga atoms forming two additional caps.

In contrast, compounds REInTe₂ are centrosymmetric, they adopt a structure with the space group Amm2. Although this can, in principle, be related to the structure of REGaTe₂ by group-subgroup relationships, the cationic modules are not shifted against each other in the indium compounds. The indium atoms are located in capped trigonal prisms that show little lone-pair influence, their environment is more symmetrical. Although these polyhedra are interconnected in a fashion that is similar to the one in REGaTe₂, the distances between the chains are not much larger than the ones within the chains so that the compound is a rare-earth indium (I) telluride rather than a telluridoindate(I) with a discrete polyanion. Still, bond valence sums correspond to RE^IIIIn^ITe^-II₂. The anionic In- or Ga-containing substructures thus show a pronounced influence on the arrangement of the cationic substructures – they interconnect similar modules is different ways. The CrB structure type may be regarded as an aristotype of both arrangements.

[1] T. Plirdpring, K. Kurosaki, A. Kosuga, T. Day, S. Firdosy, V. Ravi, G. J. Snyder, A. Harnwunggmoung, T. Sugahara, Y. Ohishi (2012) Adv. Mater. 24, 3622.
[2] S. Chatraphorn, T. Panmatarite, S. Pramatus, A. Prichavudhi, R. Kritayakirana, J.‐O. Berananda, V. Sayakanit, J. C. Woolley (1985) J. Appl. Phys. 57, 1791.
[3] A. M. Mills, A. Mar, (2001) J. Am. Chem. Soc. 123, 1151.
[4] S. P. Yatsenko, A. A. Semyannikov, B. G. Semenov, K. A. Chuntonov (1979) J. Less-Common Met. 64, 185.
[5] F. Hulliger, (1996) J. Alloys Compd. 239, 131.

Growing interest in the design of functional materials with increasingly complex crystal structures calls for a more detailed understanding of structure-property relationships in inorganic solids. Whereby functional material properties are often linked to irregular bond distances, deciphering the causal mechanisms underlying bond-length variation, and the extent to which bond lengths vary in solids, has important implications in the design of new materials and the optimization of their functional properties.

Investigation of the relation between bond-length variation and the expression of functional material properties begins with systematization of chemical-bonding behavior via large-scale bond-length dispersion analysis. Completion of the largest bond-length dispersion analysis to date for inorganic solids (177,446 reliable bond lengths hand-picked from 9210 crystal-structure refinements for oxides [1]; 6,770 bond lengths from 720 crystal-structure refinements for nitrides [2]; 33,626 bond lengths from 1832 crystal-structure refinements for chalcogenides [3]) recently enabled straightforward identification of anomalous (i.e. irregular) bonding behavior for all ions of the periodic table observed bonding to O^2-, N^3-, and S^2-/Se^2-/Te^2-. In addition to comprehensive description of bond-length variations in inorganic solids, the large amount of data on anomalous coordination environments provided by this undertaking allows (1) conclusive resolution of the causal mechanisms underlying bond-length variation in inorganic solids, and (2) quantification of the extent to which these causal mechanisms result in bond-length variation.

In a sample of 266 highly irregular coordination polyhedra covering 85 transition-metal ion configurations bonded to O^2-, the most common cause of bond-length variation is observed to be non-local bond-topological asymmetry — a widely overlooked phenomenon whose associated bond-length variation results from asymmetric patterns of a priori bond valences — followed closely by the pseudo Jahn-Teller effect (PJTE). Two new indices, and , calculated on the basis of crystallographic site, are proposed to quantify bond-length variation arising from bond-topological and crystallographic mechanisms in extended solids; is defined as the mean weighted deviation between the bond valences of a given polyhedron and that of its regular variant with equal bond lengths, while similarly quantifies the difference between a priori and observed bond valences. Bond-topological mechanisms of bond-length variation are (1) non-local bond-topological asymmetry and (2) multiple-bond formation, while crystallographic mechanisms are (3) electronic effects (e.g. vibronic mixing, lone-pair stereoactivity), and (4) crystal-structure effects (e.g. structural incommensuration).

Comprehensive bond-length dispersion analyses for inorganic nitrides [2] and chalcogenides [3] reveal several “phenomenological gaps” compared to their oxide counterparts, thus providing synthetic opportunities via the transposing of anomalously bonded coordination units bearing functional properties into new compositional and/or structural spaces. Resolving the contribution of (static) bond-topological vs (tunable) crystallographic mechanisms of bond-length variation via the and indices, combined with their spatial resolution within the coordination polyhedron and unit cell, is proposed to quantify the effective tunable extent of a functional property for a given crystal structure, e.g. via alteration of the responsible coordination unit(s). The known extent for which bond-topological and crystallographic mechanisms materialize into bond-length variations, provided by large-scale bond-length dispersion analyses, guides optimization of these properties within the constraints of physically realistic crystal structures. Such information is essential to the design of new materials with (1) increasingly complex crystal structures, and (2) superior functional properties.

[1] Gagné, O. C. & Hawthorne, F. C. (2016). Acta Cryst. B72, 602–625; Gagné, O. C. (2018). Acta Cryst. B74, 49–62; Gagné, O. C. & Hawthorne, F. C. (2018a). Acta Cryst. B74, 63–78; Gagné, O. C. & Hawthorne, F. C. (2018b). Acta Cryst. B74, 79–96; Gagné, O. C. & Hawthorne, F. C. ChemRxiv 11605698.

[2] Gagné, O. C. (2020). ChemRxiv. 11626974

[3] Gagné, O. C. et al. (2020). In preparation

This study explored the pressure dependent polymorphism of BaRhO₃ within the 7 – 22 GPa pressure range. We report the synthesis of a previously undiscovered 6H perovskite polymorph of BaRhO₃, which was stabilised between 14 – 22 GPa, below which a previously known 4H polymorph is yielded.[1] From Rietveld analysis of synchrotron X-ray powder diffraction data, the polymorph was found to crystallise in the monoclinic C2/c 6H perovskite structure, similar to the analogous BaIrO₃ 6H polymorph which is also synthesised at high pressure.[2] This data analysis also confirms a 4+ oxidation state for Rh which we believe is stabilised by the extremely high oxygen pressures accessible via high pressure synthesis. Physical property measurements and electronic structure calculations were carried out on the 4H and 6H polymorphs. Both polymorphs were found to be Pauli paramagnetic metallic oxides. Resistivity measurements confirm a metallic state for the 4H polymorph, while bulk resistivity indicates semiconductivity for the 6H polymorph. We believe this semiconducting behaviour to arise due to grain boundary effects and not to be intrinsic. High Wilson ratios of approximately 2 for either compound indicate strong electron correlations which is rationalised by strong intermetallic interactions within the Rh₂O₉ dimers. Overall this study suggests that like the neighbouring Ru, Rh oxides display physical properties driven by competing localised and itinerant electron behaviour, and that the higher oxidation states of Rh are readily accessible under high pressure, high temperature conditions.

In recent years there has been tremendous interest in perovskite-like ABX₃ hybrid frameworks, built from inorganic and organic building blocks, for their semiconducting, ferroelectric and magnetic properties. Much of the attraction in these materials lies in the well-known chemical flexibility of perovskite structures, which allows them to accommodate a wide range of cations and anions, as is well known perovskite oxides. Much of this flexibility is enhanced in inorganic-organic perovskites both with respect to their chemistry e.g. their ability to incorporate a wide range of molecular A-site cations and ligands, distortion modes and mechanical flexibility. In one key aspect, however, hybrid perovskites currently have less flexibility compared to conventional perovskites, namely the range of formal charges of cations they can incorporate. This results from the ligands in these hybrid material almost always being monovalent, which essentially restricts the A and B sites to monovalent and divalent cations, respectively.

Recent work in our group has realised a combination of monovalent and divalent ligands in perovskite-like materials via replacing HCO₂^- linker with C₂O₄^2- ligands. Most interestingly this has yielded [(CH₂)₃N]Er(HCO₂)₂(C₂O₄) and [(CH₃)₂NH₂]Er(HCO₂)₂(C₂O₄), allowing monovalent organic A-site and trivalent B-site cations to be combined for the first time in a stoichiometric ABX₃ perovskite. Our presentation will discuss the synthesis, crystal structures and magnetic properties of these materials. These exhibit A-site cation ordering up to 500 K, which will likely make related phases of interest as ferroelectrics. The greater framework flexibility in [(CH₂)₃N]Er(HCO₂)₂(C₂O₄) leads to it exhibiting significant anisotropic negative thermal expansion while the more rigid [(CH₃)₂NH₂]Er(HCO₂)₂(C₂O₄) phase does not.

The second part of our presentation will focus on the related ALn(C₂O₄)_1.5(HCO₂) (Ln = Tb-Er) phases, where we find that replacing an additional formate ligand with oxalate leads to a structure with ordered ligand vacancies. This leads to larger channels in the materials, which is likely the cause of the disorded A-site cations in these materials; ultimately the presence and nature of these A-site cations, which could not be identified crystallographically, have been confirmed by neutron and infrared spectroscopy. These two new series of materials highlight the potential to expand the flexibility of hybrid perovskite and perovskite-like materials by incorporating divalent ligands, allowing their properties to be further tailored for applications.

Rubidium tetrachloro zincate (Rb₂ZnCl₄) belongs to A₂BX₄ crystal family with the β-K₂SO₄ structure type, which are known for their ferroelectric properties and successive phase transitions. Rb₂ZnCl₄ has an orthorhombic crystal structure with Pmcn as its space group in its normal phase and goes from a normal disordered structure to incommensurately modulated structure along its c-axis at 303 K, then goes to a commensurately modulated structure around 192 K (T_c). Here we report the temperature dependent crystal structure of Rb₂ZnCl₄ in an attempt to elucidate the relation between structure and physical properties of this compound.

In the incommensurate phase the modulation wave vector is given by q = (1/3 – δ) c*, where δ is the parameter which changes with temperature, it decreases with decrease in temperature and finally becomes zero at the lock-in phase transition temperature T_c . In Rb₂ZnCl₄ the modulation wave function changes from a sinusoidal harmonic function just below the incommensurate phase transition (303K) to a strongly anharmonic function near the lock-in phase transition at T_c. The modulation function in the incommensurate phase of Rb₂ZnCl₄ is not only given by displacive modulation but also modulations of atomic displacement parameters (ADPs) and anharmonic ADPs. The structural analysis together with the lattice dynamics studies help us to understand the relation between aperiodic order and physical properties.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C- terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding of this process. Herein, we used X-ray crystallography to characterize an immature form of the main protease, which revealed major conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during the formation of the tetramer. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during maturation, which can guide the development of new inhibitors.

Atherosclerosis is a major cause of cardiovascular diseases and stroke. Oxidized low-density lipoprotein (Ox-LDL) plays a key role in the initiation and progression atherogenic process. Lectin-like ox-LDL receptor-1 (LOX-1) [1], a scavenger receptor present on vascular endothelial cells, macrophages, smooth muscle cells, and platelets, facilitates internalization of ox-LDL leading to atherosclerotic plaque formation. Existing data points towards LOX-1 as a potential target for novel anti-atherosclerosis therapy [2-3]. However, no approved therapeutics targeting LOX-1 are known. Using computational tools, we first identified a potential druggable site on the extracellular C-terminal domain (CTLD) of LOX-1. Then, using structure-based screening and molecular dynamics we have identified and short-listed molecules from chemical libraries for further validation with a combination of surface plasmon resonance, cell-based ox-LDL uptake assay, and complex crystal structures. Our data clearly shows that LOX-1 is druggable. Further studies will be performed to decipher mechanistic details of ox-LDL uptake inhibition

[1] Sawamura, T., Kume, N., Aoyama, T., Moriwaki, H., Hoshikawa, H., Aiba, Y., ... & Masaki, T. (1997). Nature. 386(6620), 73.

[2] Mehta, J. L., Sanada, N., Hu, C. P., Chen, J., Dandapat, A., Sugawara, F., ... & Sawamura, T. (2007). Circ. Res. 100(11), 1634.

[3] Pothineni, N. V. K., Karathanasis, S. K., Ding, Z., Arulandu, A., Varughese, K. I., & Mehta, J. L. (2017). J. Am. Coll. Cardiol. 69(22), 2759.

Like other photoreceptor proteins, the archaerhodopsin-3 (AR3) protein has a desensitized, inactive state which is formed in the prolonged absence of light. This dark-adapted (DA) state must be converted to the light-adapted (LA) or resting state, before the protein can generate a proton motive force. In general, receptor desensitization is commonly achieved through reversible covalent or non-covalent modifications, which typically modulate intramolecular bonding networks to stabilize a conformation that is distinct from the active resting or ground state.

Here, we present high-resolution crystal structures of the LA and DA states of AR3, solved to 1.1 Å and 1.3 Å resolution respectively [1]. We observe significant differences between the two states in the dynamics of internal water molecules that are coupled via H-bonds to the retinal Schiff base. These changes modulate the polarity of the environment surrounding the chromophore, influence the relative stability of 13-cis and all-trans retinal isomers and facilitate the conversion between the two forms. These crystal structures also allow us to gain a better understanding of the extent to which the conformation of the chromophore is coupled to the networks of internal water molecules, see Fig. 1. They highlight how minimal displacements of charged and hydrophilic groups within the low dielectric environment of the membrane can induce changes in ligand conformation and vice versa. Finally, these structures also provide high-resolution structural information that increases our understanding of the mechanism of H+ translocation by AR3, and will facilitate the design of further, more efficient AR3 mutants for applications in optogenetics.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Obesity is a global epidemic causing increased morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is a G protein-coupled receptor that plays a key role in regulation of food consumption and energy expenditure in the central nervous system, thus becoming a prime target for anti-obesity drugs. We present the cryo-EM structure of the human MC4R-G_s signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. Coupled to signaling assays and molecular dynamics simulations, the structure demonstrates the role calcium plays in receptor activation, but not inhibition. Altogether, these results fill a gap in understanding MC4R activation and provide guidelines for a structure-based design of novel and more efficient weight management drugs.

The Commander complex is a conserved regulator of intracellular trafficking. This ancient complex consists of 15 core components as well as a number of associated proteins that can be sub-divided into 3 categories: the COMMD/Coiled coil domain containing protein (CCDC) 22/CCDC93 (CCC) complex, the Retriever complex (a distant relative of the Retromer complex) and a number of associated proteins. Functionally Commander couples to Sorting Nexin 17 (SNX17) to facilitate the recycling of over 100 cell surface proteins including key receptors such as, LDLR, LRP1, p-Selectin and the amyloid precursor protein. In addition, Commander dysfunction has been linked to various disease pathologies including X-linked intellectual disability. It is therefore crucial to understand the structure, mechanism and function of this complex, an understanding that has remain largely elusive to date.

In the work presented here I have followed on from our previously published work on the structure of individual COMMD proteins [1] by reconstituting the core COMMD protein complex in vitro. The successful reconstitution of this complex using a polycistronic E. coli vector has allowed for the identification of two distinct subcomplexes of the COMMD family, a result supported by in vivo experiments conducted using quantitative mass spectrometry and a panel of COMMD knockout eHAP cell lines.

Identification of these distinct subcomplexes also allowed for the resolution of a ~3.3 Å crystal structure of COMMD subcomplex B. Revealing an intimately assembled tetramer, with interfaces along the β-strands of the highly conserved COMM domain and more subunit specific interactions between the loops on the COMM domain and the more variable helical N-terminal domain (See Figure 1). Intriguingly despite the formation of distinct subcomplexes in in vitro expression there is sufficient evidence to suggest COMMDs exist as a decameric assembly in the endogenous cellular environment. With this in mind we have used the aforementioned structure to model this assembly, revealing a geometrically perfect star assembly (See Figure 2).

[1] Healy, M. D., Hospenthal, M. K., Hall, R. J., Chandra, M., Chilton, M., Tillu, V., Chen, K., Celligoi, D. J., McDonald, F. J., Cullen, P. J., Lott, J. S., Collins, B. M., Ghai, R. (2018). eLIFE. 7, e35895.

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins that are secreted as soluble monomers and oligomerise into large circular pre-pores on the surface of cholesterol-rich membranes. Various structural changes and transitions results in insertion of β-hairpins into the lipid bilayer, forming a large β-barrel pore that results in cell lysis. We have identified a widely distributed family of bacterial proteins that share substantial structural similarity with CDCs. We have the named these proteins the “CDC-like” (CDCL) proteins, which derive from predominantly Gram-negative bacterial phyla. Many of these CDLS exist as homologous pairs. One partner of the CDCL pair, termed CDCL long, consists of four domains: three similar to CDCs and a unique fourth domain. The other partner, CDCL short, possesses three domains, all similar to CDCs. One CDCL pair, referred to as ALY long (ALY^L) and ALY short (ALY^S), originate from the species Elizabethkingia anophelis; an emerging and opportunistic pathogen of unknown virulence and transmission. We have solved the crystal structure of ALY^L, which consists of characteristic CDC domain 1 – 3 structure; however, domain 4 differs from that of CDCs significantly. In the presence of lipids, ALY^S forms a circular oligomer, while ALY^S in combination with ALY^L forms a functional pore capable of inserting into membranes. Unlike CDCs, formation of these pores is not cholesterol dependent. To determine the atomic structure of ALY pores, cryo-EM single-particle analysis is currently being pursued. Further studies include HDX-MS and lipid binding analysis to study the conformational changes and lipid binding details of pore formation. An understanding of pore formation by ALY may yield new knowledge of Elizabethkingia anophelis virulence, in addition to providing a system that could be applied to biotechnological applications.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures
may alter physiology through interaction with RXR–PXR and potentially several other nuclear receptor heterodimers.

Non-canonical DNA structures, notably G-quadruplexes and i-motifs, draw significant attention because biological evidence suggests that they play crucial roles in a variety of disease-related biological processes. G-quadruplex DNA is composed of planar guanine tetrads which are engaged in efficient p-p stacking and are further stabilized by monovalent central cation (e.g. K⁺ or Na⁺). Sequences with G-quadruplex forming potential are present in telomeres and in oncogene promoters, according to bioinformatics studies. I-motifs are intercalated hemi-protonated cytosine-rich structures formed in the C-rich sequences. Naturally, such sequences are present in the regions complimentary to the G-rich parts of genome.

In this work we have investigated nine variants of telomeric DNA with the repeat (TTGGGG)n from the organism Tetrahymena thermophila using biophysical and x-ray crystallographic studies. Biophysical characterization showed that all sequences folded into stable GQs which adopted a variety of conformations, most commonly parallel and hybrid. Native PAGE suggested that most of the sequences form multiple species in the presence of potassium. All species, but one, are monomolecular. We successfully crystallized two variants, TET25 (resolution 1.56 Å) and TET26 (three crystal forms with resolution 1.99 and 1.97 Å) and solved the structures via molecular replacement. TET25 adopted a hybrid (3+1) conformation with a four G-tetrad core, three lateral loops, one propeller loop, and 5’ snapback. TET26 fold into a parallel GQ conformation with a four G-tetrad core and three TT propeller loops. We have also investigated binding of N-methylmesoporphyrin IX (NMM) to all sequences and crystallized three variants with NMM. NMM induces parallel fold in all sequences. Both crystal structures display 5’-5’ dimers of parallel GQs with NMM bonded to the 3’ G-tetrad. NMM binds GQ with one of its faces and another NMM molecule with another. Our structural data demonstrate great plasticity of the telomeric sequence from the telomeric region of T. thermophila where small variation in the overhang length and composition leads to drastically distinct GQ structures.

I will also share our progress toward the structure an i-motif DNA from the HRAS oncogene promoter as well as the structure of repetitive DNA (CAGAGG)n from difficult-to-replicate regions of the mouse genome implicated in replication stress. Our findings have potential to contribute to the development of new and efficient anticancer therapies.

The simple elegance of the Watson-Crick DNA model reported in 1953[1] belies an underlying complexity that is central to all life. However, about thirty years then elapsed before the true complexity of DNA was revealed in high resolution crystals structures of oligonucleotides. In these structures, DNA was captured in three distinct helical forms, Z [2], B [2], and A [3], providing the first evidence for the remarkable ability of DNA to adopt different stable conformations influenced by nucleobase sequence. Since then, our understanding of the fundamental properties of DNA has been challenged further with efforts to expand the genetic code through the creation of unnatural nucleobases. These new entities include nucleobases that pair strictly through hydrophobic interactions [4, 5] and those that pair through hydrogen bonding interactions [6]. The latter nucleobases were created by Benner and coworkers and are referred to as the Artificially Expanded Genetic Information System (AEGIS) [7]. AEGIS takes advantage of alternative hydrogen bonding arrangements between Watson-Crick like pairs, a large purine-like nucleobase and a small pyridimine-like nucleobase that exclusively pair to one another rather than natural nucleobases. This concept has produced an expanded genetic code, Hachimoji DNA [8] comprising 8 letters, 4 natural and 4 unnatural, and most recently Alien DNA, comprising 4 unnatural nucleobases. These systems including unnatural base pairs (UBPs) expand the structural landscape of DNA through the creation of duplexes that do not conform to known helical forms.

In previous work, we have reported structures including up to 6 UBPs within 16 bp duplex DNA structures [9]. Our most recent work on Alien DNA includes structures with 12 UBPs of 16 base pairs (almost Alien DNA) contained within the structure captured in B-like and A-like helical forms. The B-like structures were obtained through the use of our host-guest system, which is selective for DNA sequences that can adopt helical forms that are more similar to B than A-form DNA. In this system, the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase serves as the host and a 16-mer DNA duplex as the guest [10]. Using this system, we have determined structures of numerous DNA sequences at relatively high resolution (1.6-1.8 Å) including now one with 12 UBPs. One of the almost Alien DNA sequences including 12 UBPs has crystallized in three different crystal forms, two that diffract to 1.2 Å, providing the first very detailed structural information for these UBPs including sugar conformations. These latest structures of almost Alien DNA will be presented here along with comparative analyses with natural and other less Alien DNA structures including UBPs.

[1] Watson, J.D. and F.H. Crick, (1953). Nature 171, 737-8.

[2] Wang, A.H., G.J. Quigley, F.J. Kolpak, J.L. Crawford, J.H. van Boom, G. van der Marel and A. Rich, (1979). Nature 282, 680-6.

[3] Heinemann, U., H. Lauble, R. Frank and H. Blocker, (1987). Nucleic Acids Res 15, 9531-50.

[4] Leconte, A.M., G.T. Hwang, S. Matsuda, P. Capek, Y. Hari and F.E. Romesberg, (2008). J Am Chem Soc 130, 2336-43.

[5] Hirao, I., M. Kimoto, T. Mitsui, T. Fujiwara, R. Kawai, A. Sato, Y. Harada and S. Yokoyama, (2006). Nat Methods 3, 729-35.

[6] Yang, Z., D. Hutter, P. Sheng, A.M. Sismour and S.A. Benner, (2006). Nucleic Acids Res 34, 6095-101.

[7] Sefah, K., Z. Yang, K.M. Bradley, S. Hoshika, E. Jimenez, L. Zhang, G. Zhu, S. Shanker, F. Yu, D. Turek, W. Tan and S.A. Benner, (2014). Proc Natl Acad Sci U S A 111, 1449-54.

[8] Hoshika, S., N.A. Leal, M.J. Kim, M.S. Kim, N.B. Karalkar, H.J. Kim, A.M. Bates, N.E. Watkins, Jr., H.A. SantaLucia, A.J. Meyer, S. DasGupta, J.A. Piccirilli, A.D. Ellington, J. SantaLucia, Jr., M.M. Georgiadis and S.A. Benner, (2019). Science 363, 884-887.

[9] Georgiadis, M.M., I. Singh, W.F. Kellett, S. Hoshika, S.A. Benner and N.G. Richards, (2015). J Am Chem Soc 137, 6947-55.

[10] Cote, M.L., S.J. Yohannan and M.M. Georgiadis, (2000). Acta Crystallogr D Biol Crystallogr 56, 1120-31.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a well-known endonuclease specifically targeting an AP site to initiate base excision repair. Interestingly, APE1 also bears 3′-to-5′ exonuclease activity that shows very different catalytic properties and cellular functions. The 3'-to-5' exonuclease activity of APE1 is responsible for processing matched/mismatched terminus of duplex DNA in various DNA repair pathways, as well as for nucleoside analogs removal associated with drug resistance. Due to the limited information of APE1’s exonucleolytic catalysis, its fundamental roles in various DNA repair pathways and in drug resistance are poorly understood. In addition, how APE1 exonucleolytically recognizes and processes the terminus of duplex DNA without base preference remain unclear. We determined the first two APE1-dsDNA complex structures, which displayed a dsDNA end-binding mode. Integration of our structures, biochemical assays, and molecular dynamics simulation reveals the general rules of APE1 in handling various dsDNA substrates. The DNA binding-induced RM (Arg176 and Met269) bridge formation in active site and DNA-binding modes transition between matched and mismatched termini of dsDNA compose the exquisite machinery for substrate selection, binding, and digestion. Our studies pave the way for understanding the dsDNA terminal-processing-related cellular functions and drug resistance mechanisms of APE1.

(Ref: Nat Commun. 12, 601 (2021). https://doi.org/10.1038/s41467-020-20853-2. )

Structural characterization of clinically reported missense mutations identified in BRCA1

Neha Mishra^{1, 2}, Suchita Dubey^{1, 2} Ashok K Varma^{1, 21}Advanced centre for Treatment, Research and Education in cancer, Kharghar, Navi Mumbai, Maharashtra – 410210, INDIA, ²Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai, Maharashtra – 400094, INDIA nmishra@actrec.gov.in

Germline loss of function mutations in Breast Cancer Susceptibility gene 1 (BRCA1) and Breast Cancer Susceptibility gene 2 (BRCA2) are known to be responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). BRCA1 encodes 1863 amino acids consisting of N-terminus RING domain, intrinsically disordered central DNA binding region and highly conserved two tandem repeats of BRCTs constituting the C-terminus domain (CTD)[1]. The RING domain forms a heterodimer with BRCA1-associated RING domain protein 1 (BARD1) and acts as E3- Ubiquitin ligase. However, C-terminal of the BRCA1 is known to interact with proteins containing the consensus sequences of pS-X-X-F motif to mediate different complex formation at the time of Double Strand Break Repair (DSBR). Majority of the missense mutations are found in the BRCT, RING domain and few in the central domain of BRCA1. Several studies have been performed to classify such variants of uncertain significance (VUS) in BRCA1 as pathogenic or neutral but the exact molecular mechanism of pathogenicity still remains to be deciphered[2, 3]. The aim of the present study is to evaluate the structural significance of these missense mutations located in the central and C-terminus functional domains of BRCA1 using biophysical, biochemical and in-silico tools. It was found that BRCA1 Arg866Cys in the central region, Thr1691Arg and Gly1801Asp in the BRCT domain show conformational alterations. The central non-specific DNA binding domain has also been evaluated for its conformational changes in the presence and absence of super-coiled DNA. However, a reduced DNA binding ability was observed for the mutant as compared to the wild- type protein. Further, the central region of BRCA1 has been assessed for its intrinsically disordered behaviour. Addition of 2,2,2-trifluoroethanol (TFE) led to gain of structure of the central region and therefore, less susceptibility towards proteolysis. The mutations have been characterized with the help of Size exclusion chromatography (SEC), Circular Dichroism spectroscopy, nano DSF, EMSA. With this information we would further extend our studies for protein-protein interactions of the wild type and mutant proteins using ITC, SPR and co-crystallization. The reported results will enhance our understanding towards the fundamental structural differences arising due to cancer predisposing missense mutations.

Keywords: Hereditary Breast Cancer; BRCA1; secondary structural changes; BRCT; Intrinsically disordered protein regions (IDPRs); DNA binding regions

Acknowledgement-Funding for this study was supported by Annual Scientific Fund from ACTREC-TMC. The authors thank the XRD facility at ACTREC for providing necessary support to this study.

[1] R. Roy, J. Chun, and S. N. Powell, “BRCA1 and BRCA2 : different roles in a common pathway of genome protection,” Nat. Rev. Cancer.

[2] R. W. Anantha et al., “Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance,” pp. 1–21, 2017.

[3] P. Bouwman, H. Van Der Gulden, I. Van Der Heijden, R. Drost, and C. N. Klijn, “RESEARCH ARTICLE A High-Throughput Functional Complementation Assay for Classifi cation of BRCA1 Missense Variants,” 2013.

A solid-solid phase transition (SSPT) occurs between distinguishable crystalline forms. SSPTs have been studied extensively in metallic alloys, inorganic salt or small organic molecular crystals, but much less so in biomacromolecular crystals. In particular, SSPTs involving large-scale molecular changes that are important to biological function are largely unexplored, yet may enhance our understanding of conformational space. Here, we report a systematic study of the ligand-induced SSPT in crystals of the adenine riboswitch aptamer RNA (riboA) using a combination of polarized video microscopy (PVM), solution atomic force microscopy (AFM), and time-resolved serial crystallography (TRX). The SSPT, driven by large conformational changes induced by ligand, transforms the crystal lattice from monoclinic (apo), to triclinic (intermediate lattice in a ligand-bound conformation), to orthorhombic (final bound conformational and lattice state). Using crystal structures of each state, we mapped out the changes to the crystal packing interfaces, which define the interplay between molecular conformation and crystal phase, which were corroborated by solution AFM. Using PVM to monitor changes in crystal birefringence, we characterized the kinetics of the SSPT in crystals of different sizes and ligand concentration. Together, these studies illustrate a practical approach for characterizing SSPT in biomacromolecular crystals involving large conformational changes, and provide useful spatiotemporal data for informing time-resolved crystallography experiments.

CD72 is an inhibitory co-receptor that negatively regulates B cell antigen receptor (BCR) signalling. The ligand-binding domain of CD72 at the extracellular region belongs to the C-type lectin-like domain (CTLD) superfamily. We have demonstrated that it recognizes the nuclear autoantigen Sm/RNP composed of proteins and RNA, and suppresses autoimmune diseases such as systemic lupus erythematosus [1]. The crystal structure of the ligand-binding domain of mouse CD72^a, a lupus-resistant allele, has been determined at 1.2 Å resolution. Electrostatic potential analysis of the molecular surface of CD72^a-CTLD suggest that charge distribution at the putative ligand-binding site may affect the binding affinity between CD72 and Sm/RNP.

We have determined the crystal structure of the ligand-binding domain of mouse CD72^c, a lupus-susceptible allele with reduced affinity to Sm/RNP. The obtained crystals were large enough for X-ray diffraction experiments of about 200 µm cubic, but clusters of hundreds or thousands of microcrystals (Fig. 1). Development of the micro focus X-ray beam and rapid automated data collection [2] and processing [3] systems at SPring-8 enabled us to obtain a full data set that allowed the successful structure determination and refinement at 2.5 Å resolution (Fig. 2). We took 1,400 of small-wedge (10 degree) data from 14 crystals. The data were classified based on the unit-cell dimensions or correlation coefficient between data and merged to a full data set for structure determination. Analysis of the hierarchical clustering of the small-wedge data shows that the crystal packing varies along with the c-axis direction, but no significant conformational variations were observed among the crystal structures.

The obtained structure reveals that substitutions of amino acids at the ligand-binding site do cause the inversion of the charge distribution of the molecular surface as we hypothesized. Charge repulsion between CD72^c-CTLD and strong negative charges of RNA of Sm/RNP would be the molecular mechanism of reduced affinity.

[1] Akatsu, C., Shinagawa, K., Numoto, N., Liu, Z., Ucar, A. K., Aslam, M., Phoon, S., Adachi, T., Furukawa, K., It,o N. & Tsubata, T. (2016) J. Exp. Med. 213,2691.

[2] Hirata, K., Yamashita, K., Ueno, G., Kawano, Y., Hasegawa, K., Kumasaka, T. & Yamamoto M. (2019) Acta Cryst. D75, 138.

[3] Yamashita, K., Hirata, K. & Yamamoto, M. (2018) Acta Cryst. D74, 441.

Neutron scattering is an essential element in the materials science toolkit, providing unique structural and dynamic information. It relies on an ecosystem of facilities and smaller sources, which provide access to researchers covering a vast range of scientific problems. This talk will provide an overview of the current global neutron landscape, both today and in the near future. I aim to demonstrate the scientific impact, diversity and vitality of this ecosystem, highlighting the important role that neutron scattering plays in addressing a number of societally-impactful grand challenges.

Polymer gels are noncrystalline, disordered, and soft materials, consisting of infinite polymer networks and solvent. About 30 years have passed since small-angle neutron scattering (SANS) technique was applied to structure investigations of polymer gels,¹ which was about 15 years later than its first applications to polymeric systems.² Because of random cross-linking of the gel network, the structure of a gel is inhomogeneous over a wide spatial range from a few nanometers (the mesh size) to submicrons (large clusters). This is one of the reasons why scattering studies on gels started much later. Polymer gels are inherently much complicated systems than other polymeric systems. the gel network has various types of defects, entanglements, and a broad distribution of inter-crosslink chain length. As a result, structure investigations of polymer gels with SANS were far from quantitative level, even though SANS had been contributed to advances in polymer gel science, namely, deformation,³ swelling, polyelectrolytes,⁴ volume phase transition,⁵ etc. Though it was a dream for polymer chemists to fabricate “ideal” polymer networks, consisting of uniform mesh without defects, its realization had been unsuccessful. This lecture presents an overview of the history of fabrication and characterization of inhomogeneity-free polymer gels investigated by SANS, small-angle X-ray scattering, and light scattering.

A homogeneous gel consisting of monodisperse mesh size without negligible defects, named as tetra-PEG gel, was fabricated by cross-end-coupling of tetra-arm poly(ethylene glycol) (tetra-PEG) carrying complementary end groups.⁶ The network homogeneity was confirmed by SANS and by mechanical testing.^7-9 This methodology has brought a paradigm shift in synthesis, physics, and materials science of polymer gels, and various types of gels were prepared, e.g., ion gels,¹⁰ critical cluster gels,¹¹¹²

Owing to the development of gel fabrication, it is now possible to make a tailor-made multi-component polymer network gel with unimodal mesh size, such as 2x4 gels,¹³ and DNA-cross-linked physical gels.¹⁴ This has necessitated advancement of scattering theories for polymer gels. We have developed a scattering theory for multi-component polymer networks based on random phase approximation¹⁵¹⁶ and applied it to structural analyses of DNA-cross-linked physical gels¹⁴ and deuterated/hydrogenous hetero-polymer network gels¹⁷

[1] Baumgartner, A. Picot, C. E., Ed. (1989). Molecular Basis of Polymer Networks. 42 Berlin: Springer.

[2] Ballard, D. G. H. Schelten, J.. Wignall, G. D. (1973). Eur. Polym. J. 9, 965.

[3] Mendes, E. J. Lindner, P. Buzier, M. Boue, F. Bastide, J., (1991). Phys. Rev. Lett. 66, 1595.

[4] Moussaid, A. Schosseler, F. Munch, J. P. Candau, S. J. (1993). J. Phys. II France 3, 573.

[5] Shibayama, M. Tanaka, T. Han, C. C. (1992). J. Chem. Phys. 97, 6829.

[6] Sakai, T. Matsunaga, T. Yamamoto, Y. Ito, C. Yoshida, R. Suzuki, S. (2008). Macromolecules. 41, 5379.

[7] Matsunaga, T. Sakai, T. Akagi, Y. Chung, U. Shibayama, M. (2009). Macromolecules 42, 1344.

[8] Matsunaga, T. Sakai, T. Akagi, Y. Chung, U.. Shibayama, M. (2009). Macromolecules 42, 6245.

[9] Sakai, T. Akagi, Y. Matsunaga, T. Kurakazu, M. Chung, U. Shibayama, M. (2010). Macromolecular Rapid

Communications. 31, 1954.

[10] Asai, H. Fujii, K. Ueki, T. Sakai, T. Chung, U. Watanabe, M. Han, Y. S. Kim, T. H. (2012). Macromolecules, 45,

3902.

[11] Li, X. Hirosawa, K. Sakai, T. Gilbert, E. P. Shibayama, M., (2017). Macromolecules 50, 3655.

[12] Hayashi, K. Okamoto, F. Hoshi, S. Katashima, T. Zujur, D. C.. Li, X. Shibayama, M. Gilbert, E. P. Chung, U. Ohba, S.

Oshika, T. Sakai, T., (2017). Nat. Biomed. Eng. 1, 0044.

[13] Tsuji, Y. Li, X.. Shibayama, M., (2018) Gels, 4, 50.

[14] Li, X. Ohira, M. Naito, M. Shibayama, M. (2020). in preparation.

[15] Ijichi, Y. Hashimoto, T. (1988), Polym. Comm. 29, 135.

[16] Mortensen, K. Borger, A. L. Kirkensgaard, J. J. K. Garvey, C. J. Almdal, K. Dorokhin, A. Huang, Q. Hassager, O.

(2018). Phys. Rev. Lett. 120, 207801.

[17] Ohira, M. Tsuji, Y. Watanabe, N. Morishima, K. Gilbert, E. P. Li, X.. Shibayama, M. (2019). submitted.

Mobile genetic elements (MGEs) drive evolution and adaptation throughout the tree of life. In bacteria, MGEs frequently transfer antibiotic resistance gene (ARGs) and are major drivers of resistance spreading. Their movements have been linked to the emergence of multidrug-resistant pathogens, including VRE, MRSA and ESBL, which present major public health challenges world-wide. Transposase enzymes that catalyze MGE movement, are the most abundant and most ubiquitous proteins in nature. Yet, their structure and biochemical mechanisms are poorly understood [1].

In this talk, I will present our recent discoveries on a group of transposases, which can effectively transfer ARG-carrying MGEs between diverse bacterial species in microbial communities. We have mapped the most wide-spread transposases in bacterial genomes [2] and reconstituted their molecular mechanisms [3, unpublished data]. We further characterized the biochemical steps of these MGEs and determined high-resolution crystal and cryo-EM structures of the protein-DNA assemblies involved in their transposition [4, unpublished data]. The results shed new light on the molecular strategies of transposase enzymes and elucidate how specific DNA structures enable these proteins to insert into diverse genomic sites, thus expanding ARG transfer. These insights open new possibilities for future strategies to block or prevent transposition and thus help control the spread of antibiotic resistance.

[1] Arinkin, V., Smyshlyaev, G. & Barabas, O. (2019) Curr Opin Struct Biol. 59, 168-177.

[2] Smyshlyaev, G., Bateman A. & Barabas O. (2021) Mol Syst Biol. e9880

[3] Lambertsen, L., Rubio-Cosials, A., Patil, K.R. & Barabas, O. (2018) Mol Microbiol. 107, 639-658.

[4] Rubio-Cosials, A., Schulz, E.C., Lambertsen, L., Smyshlyaev, G., Rojas-Cordova, C., Forslund, K., Karaca, E., Bebel, A., Bork, P. & Barabas, O. (2018) Cell 173, 208-220.

Recognition of DNA by proteins, both sequence and structure specific, is important in the functioning of the cell, such as in the processes of replication, transcription, and DNA repair. Twenty-five years after base flipping, a phenomenon whereby a base in normal B-DNA is swung completely out of the helix into an extrahelical position, was first observed in HhaI methyltransferase, we are still learning from and surprised by structures of protein-DNA complexes. The novel structures of the bacterium Caulobacter crescentus cell cycle-regulated DNA adenine methyltransferase (CcrM), as well as the newly discovered CamA enzyme (named for Clostridioides difficile adenine methyltransferase A) in complexes with double-stranded DNA containing their recognition sequence, will be discussed. Each of these enzymes affect their DNA substrate in a unique number of ways that are critical for their level of discrimination of their recognition DNA sequence.

CcrM in C.crescentus is responsible for maintenance methylation immediately after replication and methylates the adenine of hemimethylated GANTC. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the five base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

In the developed world, C. difficile is one of the leading causes of hospital-acquired infections. CamA-mediated methylation of the last adenine in CAAAAA is required for normal sporulation and biofilm production by this bacterium, a key step in disease transmission. Thus, selective inhibition of CamA has great therapeutic potential. CamA contains an N-terminal methyltransferase domain as well as a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These interactions provide sufficient sequence discrimination to ensure high specificity. In addition, this DNA methyltransferase has unusual features that may aide in discovery of a new selective antibiotic to combat C. difficile infection.

Knowledge acquired from these structures may also relate to other projects in our laboratory relating to mammalian epigenetics.

Enzymes with a protein kinase fold transfer phosphate from adenosine 5′-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the gamma-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell. [1]

Here we also present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation. [2]

[1] Black, M. H., Osinski, A., Gradowski, M., Servage, K. A., Pawłowski, K., Tomchick, D. R., Tagliabracci, V. S. (2019). Science 364, 787-792.

[2] Osinski, A., Black, M. H., Pawłowski, K., Chen, Z., Li, Y., Tagliabracci, V. S. (2021). bioRxiv doi: https://doi.org/10.1101/2021.04.13.439722

Results shown in this report are derived from work performed at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory. A portion of this research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Center for Cryo-EM at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We thank the Structural Biology Laboratory and the Cryo Electron Microscopy Facility at UT Southwestern Medical Center which are partially supported by grant RP170644 from the Cancer Prevention & Research Institute of Texas (CPRIT) for cryo-EM studies.

Tick-borne encephalitis virus is the cause of tick-borne encephalitis, an important arboviral disease affecting population within European and north-eastern Asian countries. There is currently no specific treatment available although it is preventable by vaccination [1, 2]. The lack of specific antiviral together with low vaccination coverage allowed the expansion of the virus within the Europe in recent years.

In the lifecycle of TBEV, NS3 helicase domain plays an essential role in viral genome replication. This domain carries out three enzymatic activities: RNA 5’-triphosphatase, RNA helicase and ATP hydrolysis. The latter activity is coupled to and provides energy for the RNA helicase activity during unwinding of the double-stranded RNA replication intermediate [3]. To understand the coupling between ATP hydrolysis and NS3 helicase activity, we determined several crystal structures of NS3 helicase, either the apo form or in complex with non-hydrolyzable ATP-analogue (AMPPNP), ADP or ADP-Pi (post-hydrolysis state). These represent structural snapshots of the key stages in ATP hydrolysis and nucleotide exchange. We also demonstrated that the ATP hydrolysis is stimulated in the presence of ssRNA but not ssDNA, both of which bind but the latter acts as a competitive inhibitor. Thus, RNA selectivity is not due to specific binding but is encoded in the coupling mechanism.

The obtained structures served as basis for molecular dynamics simulations of NS3 helicase in complex with ssRNA. RNA binding in the post-hydrolysis state leads to an allosteric change which forces opening of the ATP binding site and allows release of the resulting inorganic phosphate ion, P_i. The allosteric change is commensurate with movement of ssRNA, suggesting that this step plays a key role in the tight coupling between helicase and ATPase activities.

Quinole-dependent nitric oxide reductases (qNORs), that use Nitric oxide (NO) to generate Nitrous oxide (N₂O) as the enzymatic product in agricultural and pathogenic conditions are of major importance to food production, environment and human health. These membrane-bound enzymes strongly contribute to environmental problem at the global level (N₂O is an ozone-depleting and greenhouse gas some 300-fold more potent than CO₂) and play significant roles in survival of pathogens (qNOR from human pathogenic bacterium, Neisseria meningitidis is responsible for detoxification of NO produced to combat immune response of the host). We have determined high-resolution cryo-EM structure of active quinol-dependent nitric oxide reductases (qNOR) from Neisseria meningitidis (Nm) and Alcaligenes xylosoxidans (Ax) at 3.06Å and 3.2Å, respectively [1,2]. For NmqNOR, we have also determined the crystallographic structure at 3.15Å. All of the crystallographic structures including that of NmqNOR are monomeric [3] while both cryoEM structures showed clear dimeric arrangement. We have identified a number of factors that may trigger destabilisation of helices necessary for preserving the integrity of dimer including the use of zinc in crystallisation. Activity assay of both NmqNOR and AxqNOR in the presence of ZnCl₂ or ZnSO₄ abolished the activity. A closer examination of the NmqNOR crystallographic and cryoEM structures revealed a significant movement of TMII where one of the Zn (called Zn1) is present in the crystallographic structure and the other was at Glu498 which is pulled away from binding to Fe_B in order to ligate the second Zn (called Zn2). It is unclear if the loss of activity is due to the binding of Zn1 located near TMII or is a consequence of the removal Glu498 from the coordination of Fe_B. The mutation of Glu to Ala led to an inactive enzyme with the size exclusion chromatography indicating the major species to be a monomer. We were able to determine the cryoEM structure of this monomer (~85kD) showing that the mutation, in addition to TMII movement, causes destabilisation of additional helices (TMIX and TMX). These results and their wider implications for structure determination of membrane proteins would be discussed in the context of enzyme mechanism.

Since resolution of the first macromolecular structure, the goal of structural biology has been to link structure to function. It is now widely accepted that the latter emerges from the structural dynamics animating the macromolecule, making characterization of intermediate (and sometime excited) states of high interest to further understand molecular processes and possibly control them. With the advent of serial crystallography at X-ray free electron lasers and synchrotrons, time-resolved crystallography, performed following a specific perturbation of the crystalline system (laser excitation, substrate soak, etc), is on the verge of becoming feasible on virtually all systems opening avenues to characterize such excited and/or intermediates states. Because crystallography is an ensemble-averaged method, however, an inherent limitation is that the occupancy of intermediate states must be high enough for the “probed state” under investigation to become visible in the electron density. This is generally not the case, with “perturbed” crystals rather existing as mixtures of initial and/or final state(s) with the “probed” state. Differences in structure factor amplitudes between the reference and “perturbed” dataset can allow calculation of Fourier difference maps (F_{obs,perturbed}-F_{obs,unperturbed}), in which only the differences between the states are depicted. An even more powerful approach is to generate extrapolated structure factor amplitudes (F_{extr,perturbed}) solely describing the intermediate state and and to use these to refine its structure using conventional refinement tools. Such data processing has in the past been performed by a handful of well-experienced crystallographers with strong knowledge of existing software but still remains out of reach for a wide audience.

Here, we will present a user-friendly program, Xtrapol8, written in python and exploiting the cctbx toolbox modules, that allows the calculation of high-quality Fourier difference maps, estimation of the occupancy of the intermediate state(s) in the crystals, and generation of extrapolated structure factor amplitudes. Briefly, the program uses Bayesian statistics to weight structure factor amplitude differences [1] which are then used to generate extrapolated structure factor amplitudes for a range of possible intermediate state occupancies, with distinct weighting schemes [2, 3] (Figure 1). Based on the comparison between experimental and calculated differences, i.e. solely on experimental observations, the correct occupancy of the intermediate state is determined and its structure refined, shedding light on conformational changes not visible before. With various user-controllable parameters of which defaults are carefully chosen, the program is adapted to be used by a wide audience of structural biologists, ranging from well-experienced crystallographers to newcomers in the field. We anticipate that this program will ease and accelerate the handling of time resolved structural data, and thereby the understanding of molecular processes underlying function in a variety of proteins.

[1] Ursby, T. & Bourgeois, D. (1997), Acta Crystallogr. Sect. A, 53, 564-575.

[2] Genick, U. .,Borgstahl, G. E., Ng, K., Ren, Z., Pradervand, C., Burke, P. M., Srajer, V., Teng, T. Y., Schildkamp, W., McRee, D. E., Moffat, K. & Getzoff, E. D. (1997), Science, 275, 1471-1475.

[3] Coquelle, N., Sliwa, M., Woodhouse, J., and others, Colletier, J.-P., Schlichting, I. & Weik, M. (2018), Nature chemistry, 10, 31–37.

Over the recent decade, functional electronic materials design and discovery have shifted way from chemical-intuition-based towards data-driven synthesis and simulation. Numerous machine learning models have been developed to successfully predict materials properties and generate new crystal structures. Most existing approaches, however, rely much upon physical insights to construct handcrafted features (descriptors), which are not always readily available. For novel materials with sparse prior data and insufficient physical understanding, conventional machine learning models display limited predictability. In this talk, I will address this challenge by introducing an adaptive optimization engine for materials composition optimization, where feature engineering is not explicitly required. I then describe a use case where multi-objective Bayesian optimization with latent-variable Gaussian processes is utilized to accelerate the design of electronic metal-insulator transition compounds. Next, I will present a quantitative study on the structure-property relationship in crystal systems enabled by deep neural networks. The model which learns the structural genome could identify intrinsically similar structures in Fourier space. Finally I propose that these two methods could work harmoniously with each other towards an iterative exploration of novel functional materials.

That X-rays can affect the structure, and therefore functionality, of materials is well established. In macromolecular crystallography,

the phenomenology of ‘radiation damage’ is a mature and important field.[1] Conversely, discussions about radiation damage in small molecule crystallography are rarer and only starting to be identified.[2] X-ray-induced effects are somewhat less well studied in conventional inorganic systems, despite being implicated in a number of interesting phenomena. Examples include decomposition, conductivity enhancement, colour changes, spin-crossover, charge transfer, cell-parameter changes, crystallisation, and amorphisation.[3–5]

Cadmium cyanide, Cd(CN) ₂, is a flexible coordination polymer best studied for its strong and isotropic negative thermal expansion (NTE) effect. In this talk I will show that this NTE is actually X-ray exposure dependent: Cd(CN) ₂ contracts not only on heating but also on irradiation by X-rays.[6]

This behaviour contrasts that observed in other beam-sensitive materials, for which X-ray exposure drives lattice expansion. We call this effect ‘negative X-ray expansion’ (NXE) and suggest its origin involves an interaction between X-rays and cyanide ‘flips’; in particular, we rule out local heating as a possible mechanism.[7] Irradiation also affects the nature of a low-temperature phase

transition. Our analysis resolves discrepancies in NTE coefficients reported previously on the basis of X-ray diffraction measurements, and we establish the ‘true’ NTE behaviour of Cd(CN) ₂ across the temperature range 150–750 K. The interplay between irradiation and mechanical response in Cd(CN)₂ highlights the potential for exploiting X-ray exposure in the design of functional materials.

[1] E. F. Garman (2010) Acta Cryst. D 66, 339–351.

[2] J. Christensen, P. N. Horton, C. S. Bury, J. L. Dickerson, H. Taberman, E. F. Garman and S. J. Coles (2019) IUCrJ, 6, 703–713

[3] V. Kiryukhin, D. Casa, J. P. Hill, B. Keimer, A. Vigliante, Y. Tomioka and Y. Tokura (1997), Nature, 386, 813–815.

[4] H. Ishibashi, T. Y. Koo, Y. S. Hor, A. Borissov, P. G. Radaelli, Y. Horibe, S.-W. Cheong and V. Kiryukhin (2002), Phys. Rev. B

66, 144424

[5] M. Tu et al., (2021) Nat. Mater. 20, 93–99

[6] C. S. Coates, C. A. Murray, H. L. B. Boström, E. M. Reynolds and A. L. Goodwin (2021) Mater. Horiz.

The crystal chemistry of A^IB^IIXO₄ (A^I = Alkali ion, B^II = alkali-earth ion, X = P, V, As) is very rich and has been widely investigated, particularly the phosphate family [1]. In recent years, we have been investigated the crystal structures [2,3] and magnetic properties of some compositions within the A^IB^IIXO₄ series [4]. Besides the pure interest from a crystal chemistry point of view, the research activity related to this series of materials is driven mainly due to their ferroelectric, ferroelastic properties and possible applications as phosphors for LEDs [1, 5]. Within the rich A^IB^IIVO₄ sub-family (X = V), we have recently found a new structural type: the larnite structure with the composition NaSrVO₄ [3]. In this contribution, we are investigating its counter phosphate composition.

Despite its simple chemistry NaSrPO₄ has never been reported so far. Here, we present the synthesis, crystal structure and phase transitions of this phosphate. Surprisingly, this material exhibits a complex structure (31 atoms in the asymmetric unit-cell, Z = 10) at room temperature characterized by a strongly under bonded Na atom. This under-bonded atom is responsible for the complex and rich phase diagram as function of temperature as illustrated in Fig. 1. NaSrPO₄ exhibits 4 phase transitions between room temperature and 750°C. Besides its rich phase diagram, NaSrPO₄ exhibits a re-entrant phase transition slightly below 600°C before to reach a hexagonal paraelastic phase at high temperature. In addition, we show that the sequence of phase transitions is strongly driven by the history of the sample and several phases can be quenched at room temperature. Finally, the co-existence of Na channels within the structure with weakly bounded Na atoms makes this material a likely candidate for ionic conductivity.

[1] Isupov, V. A., (2002). Ferroelectrics 274, 203.

[2] Nénert, G., O’Meara, P. , Degen, T. (2017). Phys. Chem. Minerals 44, 455.

[3] Nénert, G., (2017). Z. Kristallogr. 232, 669.

[4] Nénert, G., et al. (2013). Inorg. Chem. 52, 9627.

[5] Choi, S., Yun, Y. J. , Kim, S. J., Jung, H.-K. (2013) Opt. Lett. 38, 1346.

Sr₂TiO₄, first member of the Ruddlesden-Popper series Sr_n+1Ti_nO_3n+1, has been long known to undergo a phase transition at 1550 °C. This transition makes the growth of single crystals of this material highly challenging, because it usually breaks the crystal into a periodic array of uneven lamellae. While the low temperature tetragonal phase is widely studied due to its close connection to the famous perovskite SrTiO₃, there is little information about the high temperature α-phase, except for an unindexed powder pattern by Drys&Trzebiatowski [1].

We stabilized the high-temperature α-Sr₂TiO₄ crystals by rapid cooling of the incongruent melt from above the liquidus temperature. The α-phase crystallizes in the orthorhombic Pna2₁ group with lattice parameters a=14.2901(5) Å b=5.8729(2) Å c=10.0872(3) Å and is isostructural to the orthorhombic forms of Sr₂VO₄ and Sr₂CrO₄ (which belong to the β-K₂SO₄ structure type). Its structure is formed by a complicated framework of large SrO_x polyhedra with tetrahedral cavities occupied by the transition metal. The tetrahedral coordination of Ti^IV makes the α-Sr₂TiO₄ quite a rare case among titanate compounds, the only other known example being the barium orthotitanate Ba₂TiO₄ [2].

However, whereas in Ba₂TiO₄ the coordination is tetrahedral in both high- and low-temperature polymorphs and the topotactic relation between the two is known, in the case of Sr₂TiO₄ a transition occurs to the layered Ruddlesden-Popper structure with octahedral titanium coordination.

In this work, we report for the first time the crystal structure of the high-temperature α-phase of Sr₂TiO₄. We elucidate the structural differences between the related compounds and discuss possible mechanism driving the structural transition.

[1] Drys, M., Trzebiatowski, W. (1957). Roczniki Chemii. 31, 489.

[2] Gunter, J., Jameson, G. (1984). Acta Cryst. C40, 207.

Magneto-caloric materials offer the possibility to design environmentally friendlier thermal management devices compared to the widely used gas-based systems [1]. These materials exhibit a change in entropy (ΔS_M) or a temperature change (ΔT_ad) when subjected to a magnetic field under isothermal or adiabatic conditions, respectively. The magnitude of change is largest about the materials Curie temperature (T_c) due to the order-disorder phase transition of the magnetic moments within the system. A suitable material must present a large magneto-caloric effect over a broad temperature span together with suitable secondary application parameters such as low heat capacity and high thermal conductivity. MnZnSb is derived from the PbFCl structure (in which the Mn sites are arranged within two-dimensional square nets), resulting in a pseudo 2D itinerant ferromagnetism which orders just above room temperature.

The first structural study reports that MnZnSb crystallises in the same anti-PbFCl-type structure as Mn₂Sb, with the tetragonal space group P4/nmm [2]. However, results from our experiments suggests that the true structure is more complex than this and has some aperiodic nature. Electron diffraction, in-house X-ray diffraction (Fig. 1(b)) and D20 neutron diffraction (Fig. 1(c)) data on powder samples all suggest that there is a small distortion of the tetragonal cell to a triclinic subgroup cell. As well as this, there appears to be incommensurate modulations in atomic positions and possibly Mn-Zn occupation (which is only seen in the neutron data). In the variable temperature neutron diffraction, we have also uncovered an unreported structural transition at ~130 K.

We have investigated the magneto-caloric properties of MnZnSb [3] using a combination of computational and experimental methods, including samples in which some Mn is substituted with Fe and Cr. Scaling analysis of the magnetic properties determines that they are second order phase transition ferromagnets and neutron diffraction has determined that the magnetic entropy change (Fig. 1(a)) is driven by the coupling of magneto-elastic strain in the square net through the magnetic transition. The primary and secondary application related properties have been measured experimentally, and the c/a parameter is identified as an accurate proxy to control the magnetic transition. Chemical substitution on the square net affords tuning of the Curie temperature over a broad temperature span between 252 and 322 K.

The epsilon phase of Fe₂O₃ (ε-Fe₂O₃, its least known polymorph) has gained considerable interest due to its intriguing properties and great application potentials. In the last few years this rare polymorph has received extraordinary attention due to its unique physical properties: it stands out for its huge coercive field (up to 2 T at room temperature), millimeter-wave ferromagnetic resonance, magneto-electric coupling, room temperature ferroelectricity, non-linear magneto-optical effect and photocatalytic activity [1-4].

ε-Fe₂O₃ presents a complex noncentrosymmetric structure (Pna2₁) with four distinct Fe sublattices: two positions in distorted octahedra (Fe1 and Fe2), one in regular octahedral environment (Fe3r), and one in distorted tetrahedral sites (Fe4t). This work examines the structural and magnetic phase transitions in ε-Fe₂O₃ nanoparticles (~20 nm) combining synchrotron X-ray and neutron diffraction measurements in the range 2-900 K. Complemented with X-ray absorption spectroscopy (XAS) and angle-dispersive X-ray diffraction under pressure up to 34 GPa.

The origin of the spin frustration was studied in the context of the rich magnetic phase diagram (with four different successive magnetic states) and its relationship with the magnetostructural transitions observed as a function of temperature. The successive magnetic transitions have been thoroughly studied in the whole temperature range, and have been fully described using the magnetic space groups approach. We have found that the spin frustration at the Fe³⁺ tetrahedral-site (Fe4t) not only is responsible for the unexpected different FIM1 (soft) and FIM2 (super-hard) commensurate ferrimagnetic phases [5], but also it is at the origin of the singular FIM2-to-ICM magnetic phase transition that disrupts the super-hard ferrimagnetic state of Pna'2₁' magnetic symmetry.

The structural evolution of ε-Fe₂O₃ is investigated across the magnetic transitions, putting the emphasis on the FIM1 (soft) to FIM2 (super-hard) phase transition. The observed coupling between structural and magnetic features explains the changes in the magnetic structures associated to the soft and super-hard phases. Puzzling changes are also observed between 150 and 100K, at the commensurate-incommensurate magnetic phase transition (FIM2-ICM) under cooling. The spiral magnetic structure previously proposed below 100 K does not match our neutron diffraction data. Incommensurate (ICM) collinear solutions compatible with neutron data are presented. This transition reduces the coercivity of ε-Fe₂O₃ (from 20 kOe to 0.8 kOe) and its ICM magnetic order (ground state) involves the formation of magnetic antiphase boundaries.

Finally, we report a polar-nonpolar structural phase transition under pressure associated to the volume collapse reported in [6]. The symmetry changes induced by pressure are fully described. The implications of this new centrosymmetric structure for understanding the mechanisms that allow the switching of the ferroelectric polarization in ε-Fe₂O₃ thin films are also analyzed.

[1] Namai, A.; Yoshikiyo, M.; Yamada, K.; Sakurai, S.; Goto, et al. (2012). Nature Commun. 3, 1035.

[2] Gich, M.; Fina, I.; Morelli, A.; Sánchez, F.; Alexe, M.; Gàzquez, J.; Fontcuberta, J.; Roig, A. (2014). Advanced Materials, 26, 4645.

[3] Xu, K.; Feng, J. S.; Liu, Z. P.; Xiang, H. J. (2018,). Physical Review Applied 9, 044011.

[4] X. Guan, L. Yao, K. Z. Rushchanskii, S. Inkinen, R. Yu, M. Ležaić, F. Sánchez, M. Gich, et al. (2020). Adv. Electron. Mater. 6, 1901134.

[5] García-Muñoz, J. L.; Romaguera, A.; Fauth, F.; Nogués, J. & Gich, M. (2017). Chemistry of Materials 29 (22), 9705.

[6] Sans, J. A., Monteseguro, V. et al. (2018). Nature Communications 9, 4554.

Keywords: ε-Fe₂O₃ , multiferroics, magnetic structures, magnetostructural coupling, nanoparticles

We acknowledge financial support from the European Research Council (ERC) under the EU Horizon 2020 programme (grant agreement No. [819623]). Also from the Spanish Ministerio de Ciencia, Innovación y Universidades (MINCIU), through Project No. RTI2018-098537-B-C21, cofunded by ERDF from EU, “Severo Ochoa” Programme for Centres of Excellence in R&D (FUNFUTURE (CEX2019-000917-S)) and MALTA Team network (RED2018-102612-T). We also acknowledge ILL and ALBA synchrotron for provision of beam time.

Simple solids such as TaS₂, NbSe₂, and CdI₂ show surprisingly complex polytypic behaviour where a number of crystalline structures can form whose unit cells differ only along their c-axis [1, 2]. This phenomena arises due to the differences in stacking sequences of the AB₂ layers along the c-axis. Although models have been developed to explain the complex phase behaviour, no model thus far has been able to account for all polytypes formed in practice [3, 4].

In this study we look at a new way of describing the structure of layered AB₂ compounds. Focusing on the layered dichalcogenides, we translate their structural degrees of freedom to a 1D model of coupled Ising chains to explain the polytypic behaviour. Our analysis suggests a family of ten ‘simplest’ ground states (Figure 1), seven of which have previously been reported. Using Monte Carlo simulations, we find that other phases identified in the literature but not expected by our model, are either describable as disordered states intermediate to our limiting phases, or mischaracterised. We proceed to show that the coupled 1D Ising chains encapsulate the behaviour of solid solutions of layered AB₂ systems, with a long term aim to link the properties of these materials to the interaction parameters relevant to the model. This phase control is an important result as it could lead to targeted design for specific properties, as structure is known to have a profound influence on materials’ properties.