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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B24-Cryo Soft X-ray Tomography
Krios II-Titan Krios II at Diamond
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Luiza
Mendonca
,
Andrew
Howe
,
James B.
Gilchrist
,
Yuewen
Sheng
,
Dapeng
Sun
,
Michael L.
Knight
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Anna-Sophia
Krebs
,
Long
Chen
,
Julika
Radecke
,
Vivian D.
Li
,
Tao
Ni
,
Ilias
Kounatidis
,
Mohamed A.
Koronfel
,
Marta
Szynkiewicz
,
Maria
Harkiolaki
,
Marisa
Martin-Fernandez
,
William
James
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 26987]
Open Access
Abstract: Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
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Jul 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
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Daming
Zhou
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Chang
Liu
,
Alexander J.
Mentzer
,
Helen M.
Ginn
,
Yuguang
Zhao
,
Helen M. E.
Duyvesteyn
,
Aekkachai
Tuekprakhon
,
Rungtiwa
Nutalai
,
Beibei
Wang
,
Guido C.
Paesen
,
Cesar
Lopez-Camacho
,
Jose
Slon-Campos
,
Bassam
Hallis
,
Naomi
Coombes
,
Kevin
Bewley
,
Sue
Charlton
,
Thomas S.
Walter
,
Donal
Skelly
,
Sheila F.
Lumley
,
Christina
Dold
,
Robert
Levin
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Derrick
Crook
,
Teresa
Lambe
,
Elizabeth
Clutterbuck
,
Sagida
Bibi
,
Amy
Flaxman
,
Mustapha
Bittaye
,
Sandra
Belij-Rammerstorfer
,
Sarah
Gilbert
,
William
James
,
Miles W.
Carroll
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Elizabeth E.
Fry
,
Juthathip
Mongkolspaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: The race to produce vaccines against SARS-CoV-2 began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK–B.1.1.7, South Africa–B.1.351 and Brazil–P.1. These variants have multiple changes in the immunodominant spike protein which facilitates viral cell entry via the Angiotensin converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K although K417N and N501Y act together against some important antibody classes. In a number of cases it would appear that convalescent and some vaccine serum offers limited protection against this variant.
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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
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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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Daming
Zhou
,
Helen M. E.
Duyvesteyn
,
Cheng-Pin
Chen
,
Chung-Guei
Huang
,
Ting-Hua
Chen
,
Shin-Ru
Shih
,
Yi-Chun
Lin
,
Chien-Yu
Cheng
,
Shu-Hsing
Cheng
,
Yhu-Chering
Huang
,
Tzou-Yien
Lin
,
Che
Ma
,
Jiandong
Huo
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Pranav
Shah
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Robert F.
Donat
,
Kerry
Godwin
,
Karen R.
Buttigieg
,
Julia A.
Tree
,
Julika
Radecke
,
Neil
Paterson
,
Piyada
Supasa
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Miles W.
Carroll
,
Javier
Gilbert-Jaramillo
,
Michael L.
Knight
,
William
James
,
Raymond J.
Owens
,
James H.
Naismith
,
Alain R.
Townsend
,
Elizabeth E.
Fry
,
Yuguang
Zhao
,
Jingshan
Ren
,
David I.
Stuart
,
Kuan-Ying A.
Huang
Diamond Proposal Number(s):
[19946, 26983]
Abstract: The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD–EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
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Jul 2020
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