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Instruments / Technical Area	Publication Details	Published
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
	Approaches to using the chameleon: robust, automated, fast-plunge cryoEM specimen preparation Talya S. Levitz , Miriam Weckener , Ivan Fong , James H. Naismith , Catherine L. Drennan , Edward J. Brignole , Daniel K. Clare , Michele C. Darrow Frontiers In Molecular Biosciences 9 DOI: 10.3389/fmolb.2022.903148 Open Access Show Abstract ▼ Abstract: The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.	Jun 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
B21-High Throughput SAXS I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM Tomasz Uchański , Simonas Masiulis , Baptiste Fischer , Valentina Kalichuk , Uriel López-Sánchez , Eleftherios Zarkadas , Miriam Weckener , Andrija Sente , Philip Ward , Alexandre Wohlkonig , Thomas Zogg , Han Remaut , James Naismith , Hugues Nury , Wim Vranken , A. Radu Aricescu , Els Pardon , Jan Steyaert Nature Methods 18, 60 - 68 DOI: 10.1038/s41592-020-01001-6 Show Abstract ▼ Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.	Jan 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
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	An internal thioester in a pathogen surface protein mediates covalent host binding Miriam Walden , John M Edwards , Aleksandra M Dziewulska , Rene Bergmann , Gerhard Saalbach , Su-Yin Kan , Ona K. Miller , Miriam Weckener , Rosemary J. Jackson , Sally L. Shirran , Catherine H. Botting , Gordon J. Florence , Manfred Rohde , Mark J. Banfield , Ulrich Schwarz-Linek Elife 4 DOI: 10.7554/eLife.06638 PMID: 26032562 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions.	Jun 2015

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