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Instruments / Technical Area	Publication Details	Published
Krios IV-Titan Krios IV at Diamond	Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins 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 Nature Communications 12 DOI: 10.1038/s41467-021-21505-9 Diamond Proposal Number(s): [20223] Open Access Show Abstract ▼ 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.	Feb 2021
B21-High Throughput SAXS I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Structures of influenza A virus RNA polymerase offer insight into viral genome replication 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 Nature 573, 287 - 290 DOI: 10.1038/s41586-019-1530-7 Diamond Proposal Number(s): [10627, 14744, 19946, 14856, 20233] Show Abstract ▼ 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.	Sep 2019
Krios I-Titan Krios I at Diamond	GABAA receptor signalling mechanisms revealed by structural pharmacology Simonas Masiulis , Rooma Desai , Tomasz Uchanski , Itziar Serna Martin , Duncan Laverty , Dimple Karia , Tomas Malinauskas , Jasenko Zivanov , Els Pardon , Abhay Kotecha , Jan Steyaert , Keith W. Miller , A. Radu Aricescu Nature 565, 454 - 459 DOI: 10.1038/s41586-018-0832-5 Show Abstract ▼ Abstract: Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.	Jan 2019
I04-Macromolecular Crystallography	A mechanism for the activation of the influenza virus transcriptase Itziar Serna Martin , Narin Hengrung , Max Renner , Jane Sharps , Mónica Martínez-alonso , Simonas Masiulis , Jonathan M. Grimes , Ervin Fodor Molecular Cell DOI: 10.1016/j.molcel.2018.05.011 Diamond Proposal Number(s): [8423] Open Access Show Abstract ▼ Abstract: Influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB1, PB2, and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. For transcription, FluPol interacts with the C-terminal domain (CTD) of RNA polymerase II (Pol II), which enables FluPol to snatch capped RNA primers from nascent host RNAs. Here, we describe the co-crystal structure of influenza C virus polymerase (FluPolC) bound to a Ser5-phosphorylated CTD (pS5-CTD) peptide. The position of the CTD-binding site at the interface of PB1, P3, and the flexible PB2 C-terminal domains suggests that CTD binding stabilizes the transcription-competent conformation of FluPol. In agreement, both cap snatching and capped primer-dependent transcription initiation by FluPolC are enhanced in the presence of pS5-CTD. Mutations of amino acids in the CTD-binding site reduce viral mRNA synthesis. We propose a model for the activation of the influenza virus transcriptase through its association with pS5-CTD of Pol II.	Jun 2018

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