I03-Macromolecular Crystallography
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Chang
Liu
,
Helen M.
Ginn
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Beibei
Wang
,
Aekkachai
Tuekprakhon
,
Rungtiwa
Nutalai
,
Daming
Zhou
,
Alexander J.
Mentzer
,
Yuguang
Zhao
,
Helen M. E.
Duyvesteyn
,
César
López-Camacho
,
Jose
Slon-Campos
,
Thomas
Walter
,
Donal
Skelly
,
Sile Ann
Johnson
,
Thomas G.
Ritter
,
Chris
Mason
,
Sue Ann
Costa Clemens
,
Felipe Gomes
Naveca
,
Valdinete
Nascimento
,
Fernanda
Nascimento
,
Cristiano
Fernandes Da Costa
,
Paola Cristina
Resende
,
Alex
Pauvolid-Correa
,
Marilda M.
Siqueira
,
Christina
Dold
,
Nigel
Temperton
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Derrick
Crook
,
Teresa
Lambe
,
Elizabeth
Clutterbuck
,
Sagida
Bibi
,
Amy
Flaxman
,
Mustapha
Bittaye
,
Sandra
Belij-Rammerstorfer
,
Sarah C.
Gilbert
,
Tariq
Malik
,
Miles W.
Carroll
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Vicky
Baillie
,
Natali
Serafin
,
Zanele
Ditse
,
Kelly
Da Silva
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Shabir
Madhi
,
Marta C.
Nunes
,
Philip
Goulder
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Abstract: SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations.
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Jun 2021
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I03-Macromolecular Crystallography
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Piyada
Supasa
,
Daming
Zhou
,
Wanwisa
Dejnirattisai
,
Chang
Liu
,
Alexander J.
Mentzer
,
Helen M.
Ginn
,
Yuguang
Zhao
,
Helen M. E.
Duyvesteyn
,
Rungtiwa
Nutalai
,
Aekkachai
Tuekprakhon
,
Beibei
Wang
,
Guido
Paesen
,
Jose
Slon-Campos
,
César
López-Camacho
,
Bassam
Hallis
,
Naomi
Coombes
,
Kevin
Bewley
,
Sue
Charlton
,
Thomas S.
Walter
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Donal
Skelly
,
Sheila F.
Lumley
,
Natalie
Baker
,
Imam
Shaik
,
Holly
Humphries
,
Kerry
Godwin
,
Nick
Gent
,
Alex
Sienkiewicz
,
Christina
Dold
,
Robert
Levin
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Paul
Klenerman
,
Derrick
Crook
,
Teresa
Lambe
,
Elizabeth
Clutterbuck
,
Sagida
Bibi
,
Amy
Flaxman
,
Mustapha
Bittaye
,
Sandra
Belij-Rammerstorfer
,
Sarah
Gilbert
,
David R.
Hall
,
Mark
Williams
,
Neil G.
Paterson
,
William
James
,
Miles W.
Carroll
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: SARS-CoV-2 has caused over 2M deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbours 9 amino-acid changes in the spike, including N501Y in the ACE2 interacting-surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterised monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.
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Feb 2021
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Wanwisa
Dejnirattisai
,
Daming
Zhou
,
Helen M.
Ginn
,
Helen M. E.
Duyvesteyn
,
Piyada
Supasa
,
James Brett
Case
,
Yuguang
Zhao
,
Thomas
Walter
,
Alexander J.
Mentzer
,
Chang
Liu
,
Beibei
Wang
,
Guido C.
Paesen
,
Jose
Slon-Campos
,
César
López-Camacho
,
Natasha M.
Kafai
,
Adam L.
Bailey
,
Rita E.
Chen
,
Baoling
Ying
,
Craig
Thompson
,
Jai
Bolton
,
Alex
Fyfe
,
Sunetra
Gupta
,
Tiong Kit
Tan
,
Javier
Gilbert-Jaramillo
,
William
James
,
Michael
Knight
,
Miles W.
Carroll
,
Donal
Skelly
,
Christina
Dold
,
Yanchun
Peng
,
Robert
Levin
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Paul
Klenerman
,
Nigel
Temperton
,
David R.
Hall
,
Mark A.
Williams
,
Neil G.
Paterson
,
Felicity
Bertram
,
C. Alistair
Siebert
,
Daniel K.
Clare
,
Andrew
Howe
,
Julika
Radecke
,
Yun
Song
,
Alain R.
Townsend
,
Kuan-Ying A.
Huang
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Michael S.
Diamond
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009, 26983]
Open Access
Abstract: Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike, and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50<0.1μg/ml) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryo-electron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Enteroviruses cause a range of human and animal diseases, some life-threatening, but there remain no licenced anti-enterovirus drugs. However, a benzene-sulfonamide derivative and related compounds have been shown recently to block infection of a range of enteroviruses by binding the capsid at a positively-charged surface depression conserved across many enteroviruses. It has also been established that glutathione is essential for the assembly of many enteroviruses, interacting with the capsid proteins to facilitate the formation of the pentameric assembly intermediate, although the mechanism is unknown. Here we show, by high resolution structure analyses of enterovirus F3, that reduced glutathione binds to the same interprotomer pocket as the benzene-sulfonamide derivative. Bound glutathione makes strong interactions with adjacent protomers, thereby explaining the underlying biological role of this druggable binding pocket and delineating the pharmacophore for potential antivirals.
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Jan 2020
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I03-Macromolecular Crystallography
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Victoria A.
Avanzato
,
Kasopefoluwa Y.
Oguntuyo
,
Marina
Escalera-Zamudio
,
Bernardo
Gutierrez
,
Michael
Golden
,
Sergei L.
Kosakovsky Pond
,
Rhys
Pryce
,
Thomas S.
Walter
,
Jeffrey
Seow
,
Katie J.
Doores
,
Oliver G.
Pybus
,
Vincent J.
Munster
,
Benhur
Lee
,
Thomas A.
Bowden
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F–specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F−nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.
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Nov 2019
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Open Access
Abstract: Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.
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Mar 2019
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Kamel
El Omari
,
Sai
Li
,
Abhay
Kotecha
,
Thomas S.
Walter
,
Eduardo A.
Bignon
,
Karl
Harlos
,
Pentti
Somerharju
,
Felix
De Haas
,
Daniel K.
Clare
,
Mika
Molin
,
Felipe
Hurtado
,
Mengqiu
Li
,
Jonathan
Grimes
,
Dennis H.
Bamford
,
Nicole D.
Tischler
,
Juha T.
Huiskonen
,
Dave I.
Stuart
,
Elina
Roine
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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Feb 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lucy C.
Walters
,
Karl
Harlos
,
Simon
Brackenridge
,
Daniel
Rozbesky
,
Jordan R.
Barrett
,
Vitul
Jain
,
Thomas S.
Walter
,
Chris A.
O’callaghan
,
Persephone
Borrow
,
Mireille
Toebes
,
Scott G.
Hansen
,
Jonah
Sacha
,
Shaheed
Abdulhaqq
,
Justin M.
Greene
,
Klaus
Früh
,
Emily
Marshall
,
Louis J.
Picker
,
E. Yvonne
Jones
,
Andrew J.
Mcmichael
,
Geraldine M.
Gillespie
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.
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Aug 2018
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Philip
Roedig
,
Helen M.
Ginn
,
Tim
Pakendorf
,
Geoff
Sutton
,
Karl
Harlos
,
Thomas S.
Walter
,
Jan
Meyer
,
Pontus
Fischer
,
Ramona
Duman
,
Ismo
Vartiainen
,
Bernd
Reime
,
Martin
Warmer
,
Aaron S.
Brewster
,
Iris D.
Young
,
Tara
Michels-Clark
,
Nicholas K.
Sauter
,
Abhay
Kotecha
,
James
Kelly
,
David J.
Rowlands
,
Marcin
Sikorsky
,
Silke
Nelson
,
Daniel S.
Damiani
,
Roberto
Alonso-Mori
,
Jingshan
Ren
,
Elizabeth E.
Fry
,
Christian
David
,
David I.
Stuart
,
Armin
Wagner
,
Alke
Meents
Abstract: We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
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Jun 2017
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I03-Macromolecular Crystallography
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Xiangxi
Wang
,
Ling
Zhu
,
Minghao
Dang
,
Zhongyu
Hu
,
Qiang
Gao
,
Shuai
Yuan
,
Yao
Sun
,
Bo
Zhang
,
Jingshan
Ren
,
Abhay
Kotecha
,
Thomas S.
Walter
,
Junzhi
Wang
,
Elizabeth
Fry
,
David I.
Stuart
,
Zihe
Rao
Abstract: Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.
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Jan 2017
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