Krios I-Titan Krios I at Diamond
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James M.
Parkhurst
,
Adam D.
Crawshaw
,
C. Alistair
Siebert
,
Maud
Dumoux
,
C. David
Owen
,
Pedro
Nunes
,
David
Waterman
,
Thomas
Glen
,
David I.
Stuart
,
James H.
Naismith
,
Gwyndaf
Evans
Open Access
Abstract: Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.
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May 2023
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E02-JEM ARM 300CF
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Xudong
Pei
,
Liqi
Zhou
,
Chen
Huang
,
Mark
Boyce
,
Judy S.
Kim
,
Emanuela
Liberti
,
Yiming
Hu
,
Takeo
Sasaki
,
Peter D.
Nellist
,
Peijun
Zhang
,
David I.
Stuart
,
Angus I.
Kirkland
,
Peng
Wang
Diamond Proposal Number(s):
[17918]
Open Access
Abstract: Advances in cryogenic transmission electron microscopy have revolutionised the determination of many macromolecular structures at atomic or near-atomic resolution. This method is based on conventional defocused phase contrast imaging. However, it has limitations of weaker contrast for small biological molecules embedded in vitreous ice, in comparison with cryo-ptychography, which shows increased contrast. Here we report a single-particle analysis based on the use of ptychographic reconstruction data, demonstrating that three dimensional reconstructions with a wide information transfer bandwidth can be recovered by Fourier domain synthesis. Our work suggests future applications in otherwise challenging single particle analyses, including small macromolecules and heterogeneous or flexible particles. In addition structure determination in situ within cells without the requirement for protein purification and expression may be possible.
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May 2023
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I03-Macromolecular Crystallography
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Aiste
Dijokaite-Guraliuc
,
Raksha
Das
,
Daming
Zhou
,
Helen M.
Ginn
,
Chang
Liu
,
Helen M. E.
Duyvesteyn
,
Jiandong
Huo
,
Rungtiwa
Nutalai
,
Piyada
Supasa
,
Muneeswaran
Selvaraj
,
Thushan I.
De Silva
,
Megan
Plowright
,
Thomas A. H.
Newman
,
Hailey
Hornsby
,
Alexander J.
Mentzer
,
Donal
Skelly
,
Thomas G.
Ritter
,
Nigel
Temperton
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Cornelius
Roemer
,
Thomas P.
Peacock
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: In November 2021 Omicron BA.1, containing a raft of new spike mutations emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or SARS-CoV-2 infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional RBD amino-acid substitutions compared to BA.2. We describe a panel of 25 potent mAbs generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titre of vaccine or BA.1, BA.2 or BA.4/5 immune serum.
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Mar 2023
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Krios III-Titan Krios III at Diamond
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Pranav N. M.
Shah
,
James B.
Gilchrist
,
Björn O.
Forsberg
,
Alister
Burt
,
Andrew
Howe
,
Shyamal
Mosalaganti
,
William
Wan
,
Julika
Radecke
,
Yuriy
Chaban
,
Geoff
Sutton
,
David I.
Stuart
,
Mark
Boyce
Diamond Proposal Number(s):
[21004]
Open Access
Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.
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Mar 2023
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I03-Macromolecular Crystallography
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Jiandong
Huo
,
Aiste
Dijokaite-Guraliuc
,
Chang
Liu
,
Raksha
Das
,
Piyada
Supasa
,
Muneeswaran
Selvaraj
,
Rungtiwa
Nutalai
,
Daming
Zhou
,
Alexander J.
Mentzer
,
Donal
Skelly
,
Thomas G.
Ritter
,
Ali
Amini
,
Sagida
Bibi
,
Sandra
Adele
,
Sile Ann
Johnson
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Megan
Plowright
,
Thomas A. H.
Newman
,
Hailey
Hornsby
,
Thushan I.
De Silva
,
Nigel
Temperton
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Andrew J.
Pollard
,
Teresa
Lambe
,
Philip
Goulder
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: Variants of SARS CoV-2 have caused successive global waves of infection. These variants, with multiple mutations in the spike protein are thought to facilitate escape from natural and vaccine-induced immunity and often increase in the affinity for ACE2. The latest variant to cause concern is BA.2.75, identified in India where it is now the dominant strain, with evidence of wider dissemination. BA.2.75 is derived from BA.2 and contains four additional mutations in the receptor binding domain (RBD). Here we perform an antigenic and biophysical characterization of BA.2.75, revealing an interesting balance between humoral evasion and ACE2 receptor affinity. ACE2 affinity for BA.2.75 is increased 9-fold compared to BA.2; there is also evidence of escape of BA.2.75 from immune serum, particularly that induced by Delta infection which may explain the rapid spread in India, where BA.2.75 is now the dominant variant. ACE2 affinity appears to be prioritised over greater escape.
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Dec 2022
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Open Access
Abstract: Echoviruses, for which there are currently no approved vaccines or drugs, are responsible for a range of human diseases, for example echovirus 11 (E11) is a major cause of serious neonatal morbidity and mortality. Decay-accelerating factor (DAF, also known as CD55) is an attachment receptor for E11. Here, we report the structure of the complex of E11 and the full-length ectodomain of DAF (short consensus repeats, SCRs, 1–4) at 3.1 Å determined by cryo-electron microscopy (cryo-EM). SCRs 3 and 4 of DAF interact with E11 at the southern rim of the canyon via the VP2 EF and VP3 BC loops. We also observe an unexpected interaction between the N-linked glycan (residue 95 of DAF) and the VP2 BC loop of E11. DAF is a receptor for at least 20 enteroviruses and we classify its binding patterns from reported DAF/virus complexes into two distinct positions and orientations, named as E6 and E11 poses. Whilst 60 DAF molecules can attach to the virion in the E6 pose, no more than 30 can attach to E11 due to steric restrictions. Analysis of the distinct modes of interaction and structure and sequence-based phylogenies suggests that the two modes evolved independently, with the E6 mode likely found earlier.
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Nov 2022
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I03-Macromolecular Crystallography
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Jiandong
Huo
,
Aiste
Dijokaite-Guraliuc
,
Rungtiwa
Nutalai
,
Raksha
Das
,
Daming
Zhou
,
Alexander J.
Mentzer
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
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Nov 2022
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I03-Macromolecular Crystallography
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Aiste
Dijokaite-Guraliuc
,
Raksha
Das
,
Rungtiwa
Nutalai
,
Daming
Zhou
,
Alexander J.
Mentzer
,
Chang
Liu
,
Piyada
Supasa
,
Susanna J.
Dunachie
,
Teresa
Lambe
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
Jiandong
Huo
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Open Access
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Nov 2022
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Krios IV-Titan Krios IV at Diamond
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Mohammad W.
Bahar
,
Veronica
Nasta
,
Helen
Fox
,
Lee
Sherry
,
Keith
Grehan
,
Claudine
Porta
,
Andrew J.
Macadam
,
Nicola J.
Stonehouse
,
David J.
Rowlands
,
Elizabeth E.
Fry
,
David I.
Stuart
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.
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Nov 2022
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Open Access
Abstract: Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using Pichia pastoris, however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using Pichia pastoris. We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV.
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Oct 2022
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