I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
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Jan 2022
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Krios I-Titan Krios I at Diamond
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Claire E.
Coupland
,
Sebastian A.
Andrei
,
T. Bertie
Ansell
,
Loic
Carrique
,
Pramod
Kumar
,
Lea
Sefer
,
Rebekka A.
Schwab
,
Eamon F. X.
Byrne
,
Els
Pardon
,
Jan
Steyaert
,
Anthony I.
Magee
,
Thomas
Lanyon-Hogg
,
Mark S. P.
Sansom
,
Edward W.
Tate
,
Christian
Siebold
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
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Dec 2021
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Krios IV-Titan Krios IV at Diamond
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Max
Renner
,
Wanwisa
Dejnirattisai
,
Loic
Carrique
,
Itziar
Serna Martin
,
Dimple
Karia
,
Serban L.
Ilca
,
Shu F.
Ho
,
Abhay
Kotecha
,
Jeremy R.
Keown
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.
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Feb 2021
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20223]
Abstract: Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
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Nov 2020
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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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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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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Haitian
Fan
,
Alexander P.
Walker
,
Loic
Carrique
,
Jeremy
Keown
,
Itziar
Serna Martin
,
Dimple
Karia
,
Jane
Sharps
,
Narin
Hengrung
,
Els
Pardon
,
Jan
Steyaert
,
Jonathan M.
Grimes
,
Ervin
Fodor
Diamond Proposal Number(s):
[10627, 14744, 19946, 14856, 20233]
Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3,4,5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication—which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7,8,9,10—remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0–4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3′ cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.
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Sep 2019
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B21-High Throughput SAXS
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Urvashi
Sharma
,
Loïc
Carrique
,
Sandrine
Vadon-Le Goff
,
Natacha
Mariano
,
Rainier-Numa
Georges
,
Frederic
Delolme
,
Peppi
Koivunen
,
Johanna
Myllyharju
,
Catherine
Moali
,
Nushin
Aghajari
,
David J. S.
Hulmes
Diamond Proposal Number(s):
[9634]
Open Access
Abstract: Fibrillar collagen molecules are synthesized as precursors, procollagens, with large propeptide extensions. While a homotrimeric form (three α1 chains) has been reported in embryonic tissues as well as in diseases (cancer, fibrosis, genetic disorders), collagen type I usually occurs as a heterotrimer (two α1 chains and one α2 chain). Inside the cell, the role of the C-terminal propeptides is to gather together the correct combination of three α chains during molecular assembly, but how this occurs for different forms of the same collagen type is so far unknown. Here, by structural and mutagenic analysis, we identify key amino acid residues in the α1 and α2 C-propeptides that determine homo- and heterotrimerization. A naturally occurring mutation in one of these alters the homo/heterotrimer balance. These results show how the C-propeptide of the α2 chain has specifically evolved to permit the appearance of heterotrimeric collagen I, the major extracellular building block among the metazoa.
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Mar 2017
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