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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure of the outer membrane transporter fema and its role in the uptake of ferric dihydro-aeruginoic acid and ferric aeruginoic acid in Pseudomonas aeruginosa Virginie Will , Lucile Moynie , Elise Si Ahmed Charrier , Audrey Le Bas , Lauriane Kuhn , Florian Volck , Johana Chicher , Hava Aksoy , Morgan Madec , Cyril Antheaume , Gaëtan L. A. Mislin , Isabelle J. Schalk Acs Chemical Biology DOI: 10.1021/acschembio.4c00820 Diamond Proposal Number(s): [33133] Show Abstract ▼ Abstract: Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. 55Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA2-Fe or DHA2-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen’s strategies for iron homeostasis.	Mar 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
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2 Katy Cornish , Jiandong Huo , Luke Jones , Parul Sharma , Joseph W. Thrush , Sahar Abdelkarim , Anja Kipar , Siva Ramadurai , Miriam Weckener , Halina Mikolajek , Sai Liu , Imogen Buckle , Eleanor Bentley , Adam Kirby , Ximeng Han , Stephen M. Laidlaw , Michelle Hill , Lauren Eyssen , Chelsea Norman , Audrey Le Bas , John Clarke , William James , James P. Stewart , Miles Carroll , James Naismith , Raymond J. Owens Open Biology 14 DOI: 10.1098/rsob.230252 Diamond Proposal Number(s): [27031] Open Access Show Abstract ▼ Abstract: The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.	Jun 2024
Krios I-Titan Krios I at Diamond	The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase, Wzz, from enterobacterial common antigen biosynthesis form a complex Miriam Weckener , Laura S. Woodward , Bradley R. Clarke , Huanting Liu , Philip N. Ward , Audrey Le Bas , David Bhella , Chris Whitfield , James H. Naismith Open Biology 13 DOI: 10.1098/rsob.220373 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.	Mar 2023
I03-Macromolecular Crystallography Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes Halina Mikolajek , Miriam Weckener , Z. Faidon Brotzakis , Jiandong Huo , Evmorfia V. Dalietou , Audrey Le Bas , Pietro Sormanni , Peter J. Harrison , Philip N. Ward , Steven Truong , Lucile Moynie , Daniel K. Clare , Maud Dumoux , Joshua Dormon , Chelsea Norman , Naveed Hussain , Vinod Vogirala , Raymond J. Owens , Michele Vendruscolo , James Naismith Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2205412119 Diamond Proposal Number(s): [27031, 27051, 29666] Open Access Show Abstract ▼ Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.	Jul 2022
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 I24-Microfocus Macromolecular Crystallography	Production and crystallization of nanobodies in complex with the receptor binding domain of the SARS-COV-2 spike protein Audrey Le Bas , Halina Mikolajek , Jiandong Huo , Chelsea Norman , Joshua Dormon , James Naismith , Raymond Owens Bio-Protocol 12 DOI: 10.21769/BioProtoc.4406 Diamond Proposal Number(s): [27031] Show Abstract ▼ Abstract: The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.	May 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19 Jiandong Huo , Halina Mikolajek , Audrey Le Bas , Jordan J. Clark , Parul Sharma , Anja Kipar , Joshua Dormon , Chelsea Norman , Miriam Weckener , Daniel K. Clare , Peter J. Harrison , Julia A. Tree , Karen R. Buttigieg , Francisco J. Salguero , Robert Watson , Daniel Knott , Oliver Carnell , Didier Ngabo , Michael J. Elmore , Susan Fotheringham , Adam Harding , Lucile Moynie , Philip N. Ward , Maud Dumoux , Tessa Prince , Yper Hall , Julian A. Hiscox , Andrew Owen , William James , Miles W. Carroll , James P. Stewart , James Naismith , Raymond Owens Nature Communications 12 DOI: 10.1038/s41467-021-25480-z Diamond Proposal Number(s): [27031] Open Access Show Abstract ▼ Abstract: SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.	Sep 2021
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2 Jiangdong Huo , Audrey Le Bas , Reinis R. Ruza , Helen M. E. Duyvesteyn , Halina Mikolajek , Tomas Malinauskas , Tiong Kit Tan , Pramila Rijal , Maud Dumoux , Philip N. Ward , Jingshan Ren , Daming Zhou , Peter J. Harrison , Miriam Weckener , Daniel K. Clare , Vinod K. Vogirala , Julika Radecke , Lucile Moynie , Yuguang Zhao , Javier Gilbert-Jaramillo , Michael L. Knight , Julia A. Tree , Karen R. Buttigieg , Naomi Coombes , Michael J. Elmore , Miles W. Carroll , Loic Carrique , Pranav N. M. Shah , William James , Alain R. Townsend , David I. Stuart , Raymond J. Owens , James H. Naismith Nature Structural & Molecular Biology 21 DOI: 10.1038/s41594-020-0469-6 Diamond Proposal Number(s): [27031, 27051] Open Access Show Abstract ▼ Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.	Jul 2020

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