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

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Gluebodies offer a route to improve crystal reliability and diversity through transferable nanobody mutations that introduce constitutive close contacts Mingda Ye , Mpho Makola , Mark W. Richards , Joseph A. Newman , Michael Fairhead , Selena G. Burgess , Zhihuang Wu , Elizabeth Maclean , Nathan D. Wright , Lizbe Koekemoer , Andrew Thompson , Gustavo Arruda Bezerra , Gangshun Yi , Huanyu Li , Victor Rangel , Dimitrios Mamalis , Hazel Aitkenhead , Benjamin G. Davis , Robert J. C. Gilbert , Katharina L. Duerr , Richard Bayliss , Opher Gileadi , Frank Von Delft Acs Central Science DOI: 10.1021/acscentsci.5c00937 Diamond Proposal Number(s): [26998] Open Access Show Abstract ▼ Abstract: Design of modular, transferable protein assemblies has broad applicability and in structural biology could help with the ever-troublesome crystallization bottleneck, including finding robustly behaved protein crystals for rapidly characterizing ligands or drug candidates or generating multiple polymorphs to illuminate diverse conformations. Nanobodies as crystallization chaperones are well-established but still unreliable, as we show here. Instead, we show an exemplar of how robust crystallization behavior can be engineered by exploring many combinations (>200) of nanobody surface mutations over several iterations. Critically, what needed testing was crystallization and diffraction quality, since target–nanobody binding affinity is decoupled from crystallizability enhancement. Our study yielded multiple polymorphs, all mediated by the same interface, with dramatically improved resolution and diffraction reliability for some mutants; we thus name them ‘Gluebodies’ (Gbs). We further demonstrate that these Gb mutations do transfer to some other targets, both for achieving robust crystallization in alternative packing forms and for establishing the ability to crystallize a key early stage readout. Since the Gb interface is evidently a favored interaction, it may be broadly applicable for modular assembly; more specifically, this work suggests that Gbs should be routinely attempted for crystallization whenever nanobodies are available.	Oct 2025
Krios I-Titan Krios I at Diamond	Covalently constrained 'Di-Gembodies' enable parallel structure solutions by cryo-EM Gangshun Yi , Dimitrios Mamalis , Mingda Ye , Loic Carrique , Michael Fairhead , Huanyu Li , Katharina L. Duerr , Peijun Zhang , David B. Sauer , Frank Von Delft , Benjamin G. Davis , Robert J. C. Gilbert Nature Chemical Biology 52 DOI: 10.1038/s41589-025-01972-7 Diamond Proposal Number(s): [20223, 21004] Open Access Show Abstract ▼ Abstract: Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.	Aug 2025
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography Krios II-Titan Krios II at Diamond	Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide Audrey Le Bas , Bradley R. Clarke , Tanisha Teelucksingh , Micah Lee , Kamel El Omari , Andrew M. Giltrap , Stephen A. Mcmahon , Hui Liu , John H. Beale , Vitaliy Mykhaylyk , Ramona Duman , Neil G. Paterson , Philip N. Ward , Peter J. Harrison , Miriam Weckener , Els Pardon , Jan Steyaert , Huanting Liu , Andrew Quigley , Benjamin G. Davis , Armin Wagner , Chris Whitfield , James H. Naismith Open Biology 15 DOI: 10.1098/rsob.240310 Diamond Proposal Number(s): [33941] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.	Jan 2025
I04-Macromolecular Crystallography	Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted Lachlan P. Deimel , Lucile Moynie , Guoxuan Sun , Viliyana Lewis , Abigail Turner , Charles J. Buchanan , Sean A. Burnap , Mikhail Kutuzov , Carolin M. Kobras , Yana Demyaneko , Shabaz Mohammed , Mathew Stracy , Weston B. Struwe , Andrew J. Baldwin , James Naismith , Benjamin G. Davis , Quentin J. Sattentau Nature Communications 15 DOI: 10.1038/s41467-024-51138-7 Open Access Show Abstract ▼ Abstract: Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.	Aug 2024
NONE-No attached Diamond beamline	Pathogen-sugar interactions revealed by universal saturation transfer analysis Charles J. Buchanan , Ben Gaunt , Peter J. Harrison , Yun Yang , Jiwei Liu , Aziz Khan , Andrew M. Giltrap , Audrey Le Bas , Philip N. Ward , Kapil Gupta , Maud Dumoux , Tiong Kit Tan , Lisa Schimaski , Sergio Daga , Nicola Picchiotti , Margherita Baldassarri , Elisa Benetti , Chiara Fallerini , Francesca Fava , Annarita Giliberti , Panagiotis I. Koukos , Matthew J. Davy , Abirami Lakshminarayanan , Xiaochao Xue , Georgios Papadakis , Lachlan P. Deimel , Virgínia Casablancas-Antràs , Timothy D. W. Claridge , Alexandre M. J. J. Bonvin , Quentin J. Sattentau , Simone Furini , Marco Gori , Jiandong Huo , Raymond J. Owens , Christiane Schaffitzel , Imre Berger , Alessandra Renieri , James H. Naismith , Andrew J. Baldwin , Benjamin G. Davis Science 479 DOI: 10.1126/science.abm3125 Open Access Show Abstract ▼ Abstract: Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.	Jun 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structure-guided discovery of a selective Mcl-1 inhibitor with cellular activity Zoltan Szlávik , Levente Ondi , Márton Csékei , Attila Paczal , Zoltán B. Szabó , Gábor Radics , James Murray , James Davidson , Ijen Chen , Ben Davis , Roderick E. Hubbard , Christopher Pedder , Pawel Dokurno , Allan Surgenor , Julia Smith , Alan Robertson , Gaetane Letoumelin-Braizat , Nicolas Cauquil , Marion Zarka , Didier Demarles , Francoise Perron-Sierra , Audrey Claperon , Frederic Colland , Olivier Geneste , András Kotschy Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b00134 Diamond Proposal Number(s): [17182, 1194, 2103] Show Abstract ▼ Abstract: Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using nuclear magnetic resonance and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compounds, and cellular activity was brought on scale by offsetting the negative charge of the anchoring carboxylate group. The obtained compounds described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compound, the chemical series leading to the identification of our more advanced compounds S63845 and S64315.	Jul 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Establishing drug discovery and identification of hit series for the anti-apoptotic proteins, Bcl-2 and Mcl-1 James B. Murray , James Davidson , Ijen Chen , Ben Davis , Pawel Dokurno , Christopher J. Graham , Richard Harris , Allan Jordan , Natalia Matassova , Christopher Pedder , Stuart Ray , Stephen D. Roughley , Julia Smith , Claire Walmsley , Yikang Wang , Neil Whitehead , Douglas S. Williamson , Patrick Casara , Thierry Le Diguarher , John Hickman , Jerome Stark , András Kotschy , Olivier Geneste , Roderick E. Hubbard Acs Omega 4, 8892 - 8906 DOI: 10.1021/acsomega.9b00611 Diamond Proposal Number(s): [671, 1194, 17182] Open Access Show Abstract ▼ Abstract: We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design Yin Yao Dong , Hua Wang , Ashley C. W. Pike , Stephen A. Cochrane , Sadra Hamedzadeh , Filip J. Wyszyński , Simon R. Bushell , Sylvain F. Royer , David A. Widdick , Andaleeb Sajid , Helena I. Boshoff , Yumi Park , Ricardo Lucas , Wei-Min Liu , Seung Seo Lee , Takuya Machida , Leanne Minall , Shahid Mehmood , Katsiaryna Belaya , Wei-Wei Liu , Amy Chu , Leela Shrestha , Shubhashish M. M. Mukhopadhyay , Claire Strain-Damerell , Rod Chalk , Nicola A. Burgess-Brown , Mervyn J. Bibb , Clifton E. Barry , Carol V. Robinson , David Beeson , Benjamin G. Davis , Elizabeth P. Carpenter Cell 175, 1045 - 1058.e16 DOI: 10.1016/j.cell.2018.10.037 Diamond Proposal Number(s): [10619, 15433, 19301] Open Access Show Abstract ▼ Abstract: Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.	Nov 2018
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Palladium-mediated enzyme activation suggests multiphase initiation of glycogenesis Matthew K. Bilyard , Henry J. Bailey , Lluís Raich , Maria A. Gafitescu , Takuya Machida , Javier Iglésias-Fernández , Seung Seo Lee , Christopher D. Spicer , Carme Rovira , Wyatt W. Yue , Benjamin G. Davis Nature 441 DOI: 10.1038/s41586-018-0644-7 Show Abstract ▼ Abstract: Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2,3,4, and mouse knockout5 and inherited human mutations6 of GYG impair glycogen synthesis. GYG acts as a ‘seed core’ for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation ‘shunt’ process using on-protein C–C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG’s use of sugar substrates. This reveals a tolerant but ‘proof-read’ mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.	Oct 2018
	A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase Javier Iglesias-Fernandez , Susan M. Hancock , Seung Seo Lee , Maola Khan , Jo Kirkpatrick , Neil J. Oldham , Katherine Mcauley , Anthony Fordham-Skelton , Carme Rovira , Benjamin G. Davis Nature Chemical Biology DOI: 10.1038/nchembio.2394 Show Abstract ▼ Abstract: SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π–sugar interaction and reveal a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.	Jun 2017

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