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33 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Multiplexing methods in dynamic protein crystallography Margaret A. Klureza , Yelyzaveta Pulnova , David Von Stetten , Robin L. Owen , Godfrey S. Beddard , Arwen R. Pearson , Briony A. Yorke DOI: 10.1016/bs.mie.2024.10.009 Show Abstract ▼ Abstract: Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.	Oct 2024
I24-Microfocus Macromolecular Crystallography	An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin Jake A. Hill , Yvonne Nyathi , Sam Horrell , David Von Stetten , Danny Axford , Robin L. Owen , Godfrey S. Beddard , Arwen R. Pearson , Helen M. Ginn , Briony Yorke Communications Chemistry 7 DOI: 10.1038/s42004-024-01163-w Diamond Proposal Number(s): [27314] Open Access Show Abstract ▼ Abstract: Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.	Apr 2024
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases Jannik Strauss , Craig Wilkinson , Keni Vidilaseris , Orquidea M. De Castro Ribeiro , Jianing Liu , James Hillier , Maximilian Wichert , Anssi M. Malinen , Bernadette Gehl , Lars J. C. Jeuken , Arwen R. Pearson , Adrian Goldman Embo Reports 215 DOI: 10.1038/s44319-023-00037-x Open Access Show Abstract ▼ Abstract: Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.	Jan 2024
	A standard descriptor for fixed-target serial crystallography Robin L. Owen , Daniele De Sanctis , Arwen R. Pearson , John H. Beale Acta Crystallographica Section D Structural Biology 79 DOI: 10.1107/S2059798323005429 Open Access Show Abstract ▼ Abstract: Fixed-target crystallography has become a widely used approach for serial crystallography at both synchrotron and X-ray free-electron laser (XFEL) sources. A plethora of fixed targets have been developed at different facilities and by various manufacturers, with different characteristics and dimensions and with little or no emphasis on standardization. These many fixed targets have good reasons for their design, shapes, fabrication materials and the presence or absence of apertures and fiducials, reflecting the diversity of serial experiments. Given this, it would be a Sisyphean task to design and manufacture a new standard fixed target that would satisfy all possible experimental configurations. Therefore, a simple standardized descriptor to fully describe fixed targets is proposed rather than a standardized device. This descriptor is a dictionary that could be read by fixed-target beamline software and straightforwardly allow data collection from fixed targets new to that beamline. The descriptor would therefore allow a much easier exchange of fixed targets between sources and facilitate the uptake of new fixed targets, benefiting beamlines, users and manufacturers. This descriptor was first presented at, and was developed following, a meeting of representatives from multiple synchrotron and XFEL sources in Hamburg in January 2023.	Aug 2023
	Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease Vasundara Srinivasan , Hévila Brognaro , Prince R. Prabhu , Edmarcia Elisa De Souza , Sebastian Günther , Patrick Y. A. Reinke , Thomas J. Lane , Helen Ginn , Huijong Han , Wiebke Ewert , Janina Sprenger , Faisal H. M. Koua , Sven Falke , Nadine Werner , Hina Andaleeb , Najeeb Ullah , Bruno Alves Franca , Mengying Wang , Angélica Luana C. Barra , Markus Perbandt , Martin Schwinzer , Christina Schmidt , Lea Brings , Kristina Lorenzen , Robin Schubert , Rafael Rahal Guaragna Machado , Erika Donizette Candido , Danielle Bruna Leal Oliveira , Edison Luiz Durigon , Stephan Niebling , Angelica Struve Garcia , Oleksandr Yefanov , Julia Lieske , Luca Gelisio , Martin Domaracky , Philipp Middendorf , Michael Groessler , Fabian Trost , Marina Galchenkova , Aida Rahmani Mashhour , Sofiane Saouane , Johanna Hakanpää , Markus Wolf , Maria Garcia Alai , Dusan Turk , Arwen R. Pearson , Henry N. Chapman , Winfried Hinrichs , Carsten Wrenger , Alke Meents , Christian Betzel Communications Biology 5 DOI: 10.1038/s42003-022-03737-7 Open Access Show Abstract ▼ Abstract: SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.	Aug 2022
I24-Microfocus Macromolecular Crystallography	Structure and diffusive dynamics of Aspartate α-decarboxylase (ADC) liganded with D-Serine in aqueous solution Tushar Raskar , Stephan Niebling , Juliette M. Devos , Briony A. Yorke , Michael Haertlein , Nils Huse , V. Trevor Forsyth , Tilo Seydel , Arwen R. Pearson Physical Chemistry Chemical Physics DOI: 10.1039/D2CP02063G Diamond Proposal Number(s): [6386] Open Access Show Abstract ▼ Abstract: Incoherent neutron spectroscopy, in combination with dynamic light scattering, was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of Escherichia coli aspartate α-decarboxylase (ADC). The X-ray crystal structure of ADC in complex with the D-Serine inhibitor was also determined, and molecular dynamics simulations were used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal that D-Serine forms hydrogen bonds with some of the active site residues, that higher order oligomers of the ADC tetramer exist on ns-ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers.	Aug 2022
I03-Macromolecular Crystallography I23-Long wavelength MX	Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility Christian M. Orr , Hayden Fisher , Xiaojie Yu , Claude H.-T. Chan , Yunyun Gao , Patrick J. Duriez , Steven G. Booth , Isabel Elliott , Tatyana Inzhelevskaya , Ian Mockridge , Christine A. Penfold , Armin Wagner , Martin J. Glennie , Ann L. White , Jonathan W. Essex , Arwen R. Pearson , Mark S. Cragg , Ivo Tews Science Immunology 7 DOI: 10.1126/sciimmunol.abm3723 Diamond Proposal Number(s): [22563] Open Access Show Abstract ▼ Abstract: Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural “switch” affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.	Jul 2022
	X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease Sebastian Gunther , Patrick Y. A. Reinke , Yaiza Fernández-García , Julia Lieske , Thomas J. Lane , Helen M. Ginn , Faisal H. M. Koua , Christiane Ehrt , Wiebke Ewert , Dominik Oberthuer , Oleksandr Yefanov , Susanne Meier , Kristina Lorenzen , Boris Krichel , Janine-Denise Kopicki , Luca Gelisio , Wolfgang Brehm , Ilona Dunkel , Brandon Seychell , Henry Gieseler , Brenna Norton-Baker , Beatriz Escudero-Pérez , Martin Domaracky , Sofiane Saouane , Alexandra Tolstikova , Thomas A. White , Anna Hänle , Michael Groessler , Holger Fleckenstein , Fabian Trost , Marina Galchenkova , Yaroslav Gevorkov , Chufeng Li , Salah Awel , Ariana Peck , Miriam Barthelmess , Frank Schluenzen , Paulraj Lourdu Xavier , Nadine Werner , Hina Andaleeb , Najeeb Ullah , Sven Falke , Vasundara Srinivasan , Bruno Alves França , Martin Schwinzer , Hévila Brognaro , Cromarte Rogers , Diogo Melo , Joanna J. Zaitseva-Doyle , Juraj Knoska , Gisel E. Peña-Murillo , Aida Rahmani Mashhour , Vincent Hennicke , Pontus Fischer , Johanna Hakanpää , Jan Meyer , Philip Gribbon , Bernhard Ellinger , Maria Kuzikov , Markus Wolf , Andrea R. Beccari , Gleb Bourenkov , David Von Stetten , Guillaume Pompidor , Isabel Bento , Saravanan Panneerselvam , Ivars Karpics , Thomas R. Schneider , Maria Marta Garcia-Alai , Stephan Niebling , Christian Günther , Christina Schmidt , Robin Schubert , Huijong Han , Juliane Boger , Diana C. F. Monteiro , Linlin Zhang , Xinyuanyuan Sun , Jonathan Pletzer-Zelgert , Jan Wollenhaupt , Christian G. Feiler , Manfred S. Weiss , Eike-Christian Schulz , Pedram Mehrabi , Katarina Karničar , Aleksandra Usenik , Jure Loboda , Henning Tidow , Ashwin Chari , Rolf Hilgenfeld , Charlotte Uetrecht , Russell Cox , Andrea Zaliani , Tobias Beck , Matthias Rarey , Stephan Günther , Dusan Turk , Winfried Hinrichs , Henry N. Chapman , Arwen R. Pearson , Christian Betzel , Alke Meents Science DOI: 10.1126/science.abf7945 Open Access Show Abstract ▼ Abstract: The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.	Apr 2021
	The HARE chip for efficient time-resolved serial synchrotron crystallography Pedram Mehrabi , Henrike Mueller-Werkmeister , Jan-Philipp Leimkohl , Hendrik Schikora , Jelena Ninkovic , Silvia Krivokuca , Ladislav Andriček , Sascha W. Epp , Darren A. Sherrell , Robin L. Owen , Arwen R. Pearson , Friedjof Tellkamp , Eike Schulz , R. J. Dwayne Miller Journal Of Synchrotron Radiation 27, 360 - 370 DOI: 10.1107/S1600577520000685 Open Access Show Abstract ▼ Abstract: Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.	Mar 2020
I24-Microfocus Macromolecular Crystallography	Directed assembly of homo-pentameric cholera toxin B-subunit proteins into higher-order structures using coiled-coil appendages James F Ross , Gemma C. Wildsmith , Michael Johnson , Daniel Hurdiss , Kristian Hollingsworth , Rebecca F. Thompson , Majid Mosayebi , Chi H. Trinh , Emanuele Paci , Arwen R. Pearson , Michael E. Webb , W. Bruce Turnbull Journal Of The American Chemical Society DOI: 10.1021/jacs.8b11480 Open Access Show Abstract ▼ Abstract: The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are pre-screened for ‘designability’, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multi-subunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. X-ray crystallography and analysis of a tubular structure has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight ‘native-state’ forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.	Mar 2019

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