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444 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Defining substrate selection by rhinoviral 2A proteinase through its crystal structure with the inhibitor zVAM.fmk Karin M. Deutschmann-Olek , Wyatt W. Yue , Gustavo A. Bezerra , Tim Skern Virology 562, 128 - 141 DOI: 10.1016/j.virol.2021.07.008 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.	Oct 2021
Krios I-Titan Krios I at Diamond	Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides Dmitry Ghilarov , Satomi Inaba-Inoue , Piotr Stepien , Feng Qu , Elizabeth Michalczyk , Zuzanna Pakosz , Norimichi Nomura , Satoshi Ogasawara , Graham Charles Walker , Sylvie Rebuffat , So Iwata , Jonathan Gardiner Heddle , Konstantinos Beis Science Advances 7 DOI: 10.1126/sciadv.abj5363 Diamond Proposal Number(s): [18659] Open Access Show Abstract ▼ Abstract: Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.	Sep 2021
I03-Macromolecular Crystallography	Optimization of an imidazo[1,2-a]pyridine series to afford highly selective type i1/2 dual Mer/Axl kinase inhibitors with in vivo efficacy William Mccoull , Scott Boyd , Martin R. Brown , Muireann Coen , Olga Collingwood , Nichola L. Davies , Ann Doherty , Gary Fairley , Kristin Goldberg , Elizabeth Hardaker , Guang He , Edward J. Hennessy , Philip Hopcroft , George Hodgson , Anne Jackson , Xiefeng Jiang , Ankur Karmokar , Anne-Laure Lainé , Nicola Lindsay , Yumeng Mao , Roshini Markandu , Lindsay Mcmurray , Neville Mclean , Lorraine Mooney , Helen Musgrove , J. Willem M. Nissink , Alexander Pflug , Venkatesh Pilla Reddy , Philip B. Rawlins , Emma Rivers , Marianne Schimpl , Graham F. Smith , Sharon Tentarelli , Jon Travers , Robert I. Troup , Josephine Walton , Cheng Wang , Stephen Wilkinson , Beth Williamson , Jon Winter-Holt , Dejian Yang , Yuting Zheng , Qianxiu Zhu , Paul D. Smith Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.1c00920 Show Abstract ▼ Abstract: Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.	Sep 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	2-mercaptomethyl thiazolidines (MMTZs) inhibit all metallo-β-lactamase classes by maintaining a conserved binding mode Philip Hinchliffe , Diego M. Moreno , Maria-Agustina Rossi , Maria F. Mojica , Veronica Martinez , Valentina Villamil , Brad Spellberg , George L. Drusano , Claudia Banchio , Graciela Mahler , Robert A. Bonomo , Alejandro J. Vila , James Spencer Acs Infectious Diseases DOI: 10.1021/acsinfecdis.1c00194 Diamond Proposal Number(s): [12342, 17212] Show Abstract ▼ Abstract: Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of “cross-class” MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.	Aug 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition Yuliana Yosaatmadja , Hannah T. Baddock , Joseph A. Newman , Marcin Bielinski , Angeline E. Gavard , Shubhashish M. M. Mukhopadhyay , Adam a. Dannerfjord , Christopher J. Schofield , Peter j. Mchugh , Opher Gileadi Nucleic Acids Research 44 DOI: 10.1093/nar/gkab693 Diamond Proposal Number(s): [19301] Open Access Show Abstract ▼ Abstract: Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.	Aug 2021
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A topological switch in CFTR modulates channel activity and sensitivity to unfolding Daniel Scholl , Maud Sigoillot , Marie Overtus , Rafael Colomer Martinez , Chloé Martens , Yiting Wang , Els Pardon , Toon Laeremans , Abel Garcia-Pino , Jan Steyaert , David N. Sheppard , Jelle Hendrix , Cedric Govaerts Nature Chemical Biology 245 DOI: 10.1038/s41589-021-00844-0 Diamond Proposal Number(s): [12718, 9426] Show Abstract ▼ Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase Joseph A. Newman , Alice Douangamath , Setayesh Yadzani , Yuliana Yosaatmadja , Anthony Aimon , Jose Brandao-Neto , Louise Dunnett , Tyler Gorrie-Stone , Rachael Skyner , Daren Fearon , Matthieu Schapira , Frank Von Delft , Opher Gileadi Nature Communications 12 DOI: 10.1038/s41467-021-25166-6 Diamond Proposal Number(s): [26998] Open Access Show Abstract ▼ Abstract: There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.	Aug 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis Patrick Rabe , Jos J. A. G. Kamps , Kyle D. Sutherlin , James D. S. Linyard , Pierre Aller , Cindy C. Pham , Mikako Makita , Ian Clifton , Michael A. Mcdonough , Thomas M. Leissing , Denis Shutin , Pauline A. Lang , Agata Butryn , Jurgen Brem , Sheraz Gul , Franklin D. Fuller , In-Sik Kim , Mun Hon Cheah , Thomas Fransson , Asmit Bhowmick , Iris D. Young , Lee O'Riordan , Aaron S. Brewster , Ilaria Pettinati , Margaret Doyle , Yasumasa Joti , Shigeki Owada , Kensuke Tono , Alexander Batyuk , Mark S. Hunter , Roberto Alonso-Mori , Uwe Bergmann , Robin L. Owen , Nicholas K. Sauter , Timothy D. W. Claridge , Carol V. Robinson , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville , Christopher J. Schofield Science Advances 7 DOI: 10.1126/sciadv.abh0250 Diamond Proposal Number(s): [23459, 19458] Open Access Show Abstract ▼ Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.	Aug 2021
B24-Cryo Soft X-ray Tomography	Correlative imaging using super‐resolution fluorescence microscopy and soft X‐ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development Chidinma A. Okolo , Archana Jadhav , Patrick Phillips , Maud Dumoux , Amanda A. Mcmurray , Vishwas D. Joshi , Claire Pizzey , Maria Harkiolaki Journal Of Microscopy DOI: 10.1111/jmi.13054 Diamond Proposal Number(s): [24622] Show Abstract ▼ Abstract: Active Virosomes (AVs) are derivatives of viruses, broadly similar to ‘parent’ pathogens, with an outer envelope that contains a bespoke genome coding for 4–5 viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial “anti-viral” immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	The X-ray structure of L -threonine dehydrogenase from the common hospital pathogen Clostridium difficile Eyram Adjogatse , Josh Bennett , Jingxu Guo , Peter T. Erskine , Steve P. Wood , Brendan W. Wren , Jonathan B. Cooper Acta Crystallographica Section F Structural Biology Communications 77, 269 - 274 DOI: 10.1107/S2053230X21007135 Diamond Proposal Number(s): [7131] Show Abstract ▼ Abstract: In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD+ to oxidize its substrate to 2-amino-3-ketobutyrate. The absence of a functional TDH gene in humans suggests that inhibitors of this enzyme may have therapeutic potential against pathogens which are reliant on this enzyme. Here, TDH from Clostridium difficile has been cloned and overexpressed, and the X-ray structure of the apoenzyme form has been determined at 2.6 Å resolution.	Aug 2021

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