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
,
Dave 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
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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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Jiandong
Huo
,
Yuguang
Zhao
,
Jingshan
Ren
,
Daming
Zhou
,
Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Pranav N. M.
Shah
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Naomi
Coombes
,
Kevin R.
Bewley
,
Julia A.
Tree
,
Julika
Radecke
,
Neil
Paterson
,
Piyasa
Supasa
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Miles
Carroll
,
Alain
Townsend
,
Elizabeth E.
Fry
,
Raymond J.
Owens
,
David I.
Stuart
Diamond Proposal Number(s):
[19946, 26983]
Open Access
Abstract: There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognises angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.
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Jun 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Justina
Briliūtė
,
Paulina A.
Urbanowicz
,
Ana S.
Luis
,
Arnaud
Basle
,
Neil
Paterson
,
Osmond
Rebello
,
Jenifer
Hendel
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Eric C.
Martens
,
Daniel I. R.
Spencer
,
David N.
Bolam
,
Lucy I.
Crouch
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.
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Jun 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Graeme
Winter
,
Richard J.
Gildea
,
Neil G.
Paterson
,
John
Beale
,
Markus
Gerstel
,
Danny
Axford
,
Melanie
Vollmar
,
Katherine E.
Mcauley
,
Robin L.
Owen
,
Ralf
Flaig
,
Alun W.
Ashton
,
David
Hall
Open Access
Abstract: Strategies for collecting X-ray diffraction data have evolved alongside beamline hardware and detector developments. The traditional approaches for diffraction data collection have emphasised collecting data from noisy integrating detectors (i.e. film, image plates and CCD detectors). With fast pixel array detectors on stable beamlines, the limiting factor becomes the sample lifetime, and the question becomes one of how to expend the photons that your sample can diffract, i.e. as a smaller number of stronger measurements or a larger number of weaker data. This parameter space is explored via experiment and synthetic data treatment and advice is derived on how best to use the equipment on a modern beamline. Suggestions are also made on how to acquire data in a conservative manner if very little is known about the sample lifetime.
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Mar 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14757]
Abstract: Second harmonic generation (SHG) microscopy is widely used to image collagen fiber microarchitecture due to its high spatial resolution, optical sectioning capabilities and relatively nondestructive sample preparation. Quantification of SHG images requires sensitive methods to capture fiber alignment. This article presents a two‐dimensional discrete Fourier transform (DFT)–based method for collagen fiber structure analysis from SHG images. The method includes integrated periodicity plus smooth image decomposition for correction of DFT edge discontinuity artefact, avoiding the loss of peripheral image data encountered with more commonly used windowing methods. Outputted parameters are as follows: the collagen fiber orientation distribution, aligned collagen content and the degree of collagen fiber dispersion along the principal orientation. We demonstrate its application to determine collagen microstructure in the human optic nerve head, showing its capability to accurately capture characteristic structural features including radial fiber alignment in the innermost layers of the bounding sclera and a circumferential collagen ring in the mid‐stromal tissue. Higher spatial resolution rendering of individual lamina cribrosa beams within the nerve head is also demonstrated. Validation of the method is provided in the form of correlative results from wide‐angle X‐ray scattering and application of the presented method to other fibrous tissues.
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Jan 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11316, 14757]
Abstract: Purpose: We aimed to characterize any bulk changes in posterior scleral collagen fibril bundle architecture in human eyes with high myopia.
Methods: Wide-angle X-ray scattering (WAXS) was employed to map collagen orientation at 0.5 mm × 0.5 mm spatial intervals across the posterior sclera of seven non-myopic human eyes and three eyes with high myopia (>6D of refractive error). At each sampled point, WAXS provided thickness-averaged measures of the angular distribution of preferentially aligned collagen fibrils within the tissue plane and the anisotropic proportion (the ratio of preferentially aligned to total collagen scatter).
Results: Non-myopic specimens featured well-conserved microstructural features, including strong uniaxial collagen alignment along the extraocular muscle insertion sites of the mid-posterior sclera and a highly anisotropic annulus of collagen circumscribing the nerve head in the peripapillary sclera. All three myopic specimens exhibited notable alterations in the peripapillary sclera, including a partial loss of circumferential collagen alignment and a redistribution of the normally observed regional pattern of collagen anisotropic proportion. Linear mixed-model analysis indicated that the mean fiber angle deviation from the circumferential orientation in the peripapillary sclera of highly myopic eyes (23.9° ± 18.2) was statistically significantly higher than that of controls (17.9° ± 12.0; p<0.05).
Conclusions: Bulk alterations in the normal posterior scleral collagen microstructure occur in human eyes with high myopia. These changes could reflect remodeling of the posterior sclera during axial lengthening and/or a mechanical adaption to tissue stresses induced by fluid pressure or eye movements that may be exacerbated in enlarged eyes.
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Dec 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475]
Open Access
Abstract: The cell surface of most Gram-negative bacteria contains lipopolysaccharide that is essential for their viability and drug resistance. A 134-kDa protein complex LptB2FG is unique among ATP-binding cassette transporters because it extracts lipopolysaccharide from the external leaflet of the inner membrane and propels it along a filament that extends across the periplasm to directly deliver lipopolysaccharide into the external leaflet of the outer membrane. Here we report the crystal structure of the lipopolysaccharide transporter LptB2FG from Klebsiella pneumoniae, in which both LptF and LptG are composed of a β-jellyroll-like periplasmic domain and six α-helical segments in the transmembrane domain. LptF and LptG form a central cavity containing highly conserved hydrophobic residues. Structural and functional studies suggest that LptB2FG uses an alternating lateral access mechanism to extract lipopolysaccharide and traffic it along the hydrophobic cavity toward the transporter’s periplasmic domains.
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Aug 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Philip
Hinchliffe
,
Qiu E.
Yang
,
Edward
Portal
,
Tom
Young
,
Hui
Li
,
Catherine L.
Tooke
,
Maria J.
Carvalho
,
Neil G.
Paterson
,
Jurgen
Brem
,
Pannika R.
Niumsup
,
Uttapoln
Tansawai
,
Lei
Lei
,
Mei
Li
,
Zhangqi
Shen
,
Yang
Wang
,
Christopher J.
Schofield
,
Adrian J
Mulholland
,
Jianzhong
Shen
,
Natalie
Fey
,
Timothy R.
Walsh
,
James
Spencer
Diamond Proposal Number(s):
[12342]
Open Access
Abstract: The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.
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Jan 2017
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