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54 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
	Transient transfection and expression of eukaryotic membrane proteins in Expi293F cells and their screening on a small scale: Application for structural studies Ganna O. Krasnoselska , Maud Dumoux , Nadisha Gamage , Harish Cheruvara , James Birch , Andrew Quigley , Raymond J. Owens DOI: 10.1007/978-1-0716-1406-8_5 Show Abstract ▼ Abstract: Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.	May 2021
B21-High Throughput SAXS	Human DDX17 unwinds rift valley fever virus non-coding RNAs Corey R. Nelson , Tyler Mrozowich , Sean M. Park , Simmone D’souza , Amy Henrickson , Justin R. J. Vigar , Hans-Joachim Wieden , Raymond J. Owens , Borries Demeler , Trushar R. Patel International Journal Of Molecular Sciences 22 DOI: 10.3390/ijms22010054 Diamond Proposal Number(s): [22113] Open Access Show Abstract ▼ Abstract: Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135–555 interacting with and unwinding RVFV non-coding regions.	Jan 2021
	A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses Tiong Kit Tan , Pramila Rijal , Rolle Rahikainen , Anthony H. Keeble , Lisa Schimanski , Saira Hussain , Ruth Harvey , Jack W. P. Hayes , Jane C. Edwards , Rebecca K. Mclean , Veronica Martini , Miriam Pedrera , Nazia Thakur , Carina Conceicao , Isabelle Dietrich , Holly Shelton , Anna Ludi , Ginette Wilsden , Clare Browning , Adrian K. Zagrajek , Dagmara Bialy , Sushant Bhat , Phoebe Stevenson-Leggett , Philippa Hollinghurst , Matthew Tully , Katy Moffat , Chris Chiu , Ryan Waters , Ashley Gray , Mehreen Azhar , Valerie Mioulet , Joseph Newman , Amin S. Asfor , Alison Burman , Sylvia Crossley , John A. Hammond , Elma Tchilian , Bryan Charleston , Dalan Bailey , Tobias J. Tuthill , Simon P. Graham , Helen M. E. Duyvesteyn , Tomas Malinauskas , Jiandong Huo , Julia A. Tree , Karen R. Buttigieg , Raymond J. Owens , Miles W. Carroll , Rodney S. Daniels , John W. Mccauley , David I. Stuart , Kuan-Ying A. Huang , Mark Howarth , Alain R. Townsend Nature Communications 12 DOI: 10.1038/s41467-020-20654-7 Open Access Show Abstract ▼ Abstract: There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.	Jan 2021
I24-Microfocus Macromolecular Crystallography	The structure-function relationship of oncogenic LMTK3 Angeliki Ditsiou , Chiara Cilibrasi , Nikiana Simigdala , Athanasios Papakyriakou , Leanne Milton-Harris , Viviana Vella , Joanne E. Nettleship , Jae Ho Lo , Shivani Soni , Goar Smbatyan , Panagiota Ntavelou , Teresa Gagliano , Maria Chiara Iachini , Sahir Khurshid , Thomas Simon , Lihong Zhou , Storm Hassell-Hart , Philip Carter , Laurence H. Pearl , Robin L. Owen , Raymond J. Owens , S. Mark Roe , Naomi E. Chayen , Heinz-Josef Lenz , John Spencer , Chrisostomos Prodromou , Apostolos Klinakis , Justin Stebbing , Georgios Giamas Science Advances 6 DOI: 10.1126/sciadv.abc3099 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.	Nov 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient 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 Nature Structural & Molecular Biology 20 DOI: 10.1038/s41594-020-0480-y Diamond Proposal Number(s): [19946, 26983] Show Abstract ▼ 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.	Jul 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2 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 Nature Structural & Molecular Biology 21 DOI: 10.1038/s41594-020-0469-6 Diamond Proposal Number(s): [27031, 27051] Open Access Show Abstract ▼ 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.	Jul 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Neutralisation of SARS-CoV-2 by destruction of the prefusion Spike 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 Cell Host & Microbe DOI: 10.1016/j.chom.2020.06.010 Diamond Proposal Number(s): [19946, 26983] Open Access Show Abstract ▼ 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.	Jun 2020
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography VMXi-Versatile Macromolecular Crystallography in situ	Molecular basis for GTP recognition by light‐activated guanylate cyclase RhGC Agata Butryn , Hadeeqa Raza , Heather Rada , Isabel Moraes , Raymond J. Owens , Allen M. Orville The Febs Journal DOI: 10.1111/febs.15167 Diamond Proposal Number(s): [16818, 19737, 19485] Open Access Show Abstract ▼ Abstract: Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signaling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalyzed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin‐GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.	Dec 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus Jingshan Ren , Joanne E. Nettleship , Gemma Harris , William Mwangi , Nahid Rhaman , Clare Grant , Abhay Kotecha , Elizabeth Fry , Bryan Charleston , David I. Stuart , John Hammond , Raymond J. Owens Molecular Immunology 112, 123 - 130 DOI: 10.1016/j.molimm.2019.04.026 Diamond Proposal Number(s): [10627, 14744] Open Access Show Abstract ▼ Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.	Aug 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs Juliana Roverta Torini , Adriano De Freitas Fernandes , Vitor Hugo Balasco Serrao , Larissa Romanello , Louise Bird , Joanne E. Nettleship , Raymond J. Owens , Jose Brandao-neto , Ana Eliza Zeraik , Ricardo Demarco , Humberto D'muniz Pereira Molecular And Biochemical Parasitology 231 DOI: 10.1016/j.molbiopara.2019.111187 Diamond Proposal Number(s): [5073, 14493] Show Abstract ▼ Abstract: Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.	Jul 2019

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