Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16637]
Open Access
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.
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Mar 2023
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I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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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
Diamond Proposal Number(s):
[27031, 27051, 29666]
Open Access
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.
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Jul 2022
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NONE-No attached Diamond beamline
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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
Open Access
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.
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Jun 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[27031]
Open Access
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.
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Sep 2021
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein “Wzx-Wzy” system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
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Jul 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
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.
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Jan 2021
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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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
Diamond Proposal Number(s):
[27031, 27051]
Open Access
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.
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Jul 2020
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I02-Macromolecular Crystallography
Data acquisition
Detectors
Diagnostics
Health Physics
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Open Access
Abstract: DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.
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Jun 2016
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I04-Macromolecular Crystallography
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Steven
Johnson
,
Lionel
Tan
,
Stijn
Van Der Veen
,
Joseph
Caesar
,
Elena
Goicoechea De Jorge
,
Rachel J.
Harding
,
Xilian
Bai
,
Rachel M.
Exley
,
Philip N.
Ward
,
Nicola
Ruivo
,
Kaushali
Trivedi
,
Elspeth
Cumber
,
Rhian
Jones
,
Luke
Newham
,
David
Staunton
,
Rafael
Ufret-Vincenty
,
Ray
Borrow
,
Matthew C.
Pickering
,
Susan
Lea
,
Christoph M.
Tang
Open Access
Abstract: Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.
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Oct 2012
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