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12 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Exploring the effect of structure-based scaffold hopping on the inhibition of coxsackievirus A24v transduction by pentavalent N-acetylneuraminic acid conjugates Emil Johansson , Rémi Caraballo , Daniel L. Hurdiss , Nitesh Mistry , C. David Andersson , Rebecca F. Thompson , Neil A. Ranson , Georg Zocher , Thilo Stehle , Niklas Arnberg , Mikael Elofsson International Journal Of Molecular Sciences 22 DOI: 10.3390/ijms22168418 Diamond Proposal Number(s): [19691] Open Access Show Abstract ▼ Abstract: Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.	Aug 2021
I24-Microfocus Macromolecular Crystallography	The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding Paul White , Samuel F. Haysom , Matthew G. Iadanza , Anna J. Higgins , Jonathan M. Machin , James M. Whitehouse , Jim E. Horne , Bob Schiffrin , Charlotte Carpenter-Platt , Antonio N. Calabrese , Kelly M. Storek , Steven T. Rutherford , David J. Brockwell , Neil A. Ranson , Sheena E. Radford Nature Communications 12 DOI: 10.1038/s41467-021-24432-x Diamond Proposal Number(s): [19248] Open Access Show Abstract ▼ Abstract: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.	Jul 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Insights into SusCD-mediated glycan import by a prominent gut symbiont Declan A. Gray , Joshua B. R. White , Abraham O. Oluwole , Parthasarathi Rath , Amy J. Glenwright , Adam Mazur , Michael Zahn , Arnaud Basle , Carl Morland , Sasha L. Evans , Alan Cartmell , Carol V. Robinson , Sebastian Hiller , Neil A. Ranson , David N. Bolam , Bert Van Den Berg Nature Communications 12 DOI: 10.1038/s41467-020-20285-y Diamond Proposal Number(s): [13587, 18598] Open Access Show Abstract ▼ Abstract: In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.	Jan 2021
Krios II-Titan Krios II at Diamond	Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels David J. K. Swainsbury , Pu Qian , Philip J. Jackson , Kaitlyn M. Faries , Dariusz M. Niedzwiedzki , Elizabeth C. Martin , David A. Farmer , Lorna A. Malone , Rebecca F. Thompson , Neil A. Ranson , Daniel P. Canniffe , Mark J. Dickman , Dewey Holten , Christine Kirmaier , Andrew Hitchcock , C. Neil Hunter Science Advances 7 DOI: 10.1126/sciadv.abe2631 Diamond Proposal Number(s): [19832] Open Access Show Abstract ▼ Abstract: The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.	Jan 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis Mariusz Madej , Joshua B. R. White , Zuzanna Nowakowska , Shaun Rawson , Carsten Scavenius , Jan J. Enghild , Grzegorz P. Bereta , Karunakar Pothula , Ulrich Kleinekathoefer , Arnaud Basle , Neil A. Ranson , Jan Potempa , Bert Van Den Berg Nature Microbiology 25 DOI: 10.1038/s41564-020-0716-y Diamond Proposal Number(s): [13587] Show Abstract ▼ Abstract: Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a ‘pedal bin’ mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.	May 2020
Krios I-Titan Krios I at Diamond	Plant-made nervous necrosis virus-like particles protect fish against disease Johanna Marsian , Daniel L. Hurdiss , Neil A. Ranson , Anneli Ritala , Richard Paley , Irene Cano , George P. Lomonossoff Frontiers In Plant Science 10 DOI: 10.3389/fpls.2019.00880 Diamond Proposal Number(s): [13452] Open Access Show Abstract ▼ Abstract: Virus-like particles (VLPs) of the fish virus, Atlantic Cod Nervous necrosis virus (ACNNV), were successfully produced by transient expression of the coat protein in Nicotiana benthamiana plants. VLPs could also be produced in transgenic tobacco BY-2 cells. The protein extracted from plants self-assembled into T = 3 particles, that appeared to be morphologically similar to previously analyzed NNV VLPs when analyzed by high resolution cryo-electron microscopy. Administration of the plant-produced VLPs to sea bass (Dicentrarchus labrax) showed that they could protect the fish against subsequent virus challenge, indicating that plant-produced vaccines may have a substantial future role in aquaculture.	Jul 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids Oliver W. Bayfield , Evgeny Klimuk , Dennis C. Winkler , Emma L. Hesketh , Maria Chechik , Naiqian Cheng , Eric C. Dykeman , Leonid Minakhin , Neil A. Ranson , Konstantin Severinov , Alasdair C. Steven , Alfred A. Antson Proceedings Of The National Academy Of Sciences 116, 3556 - 3561 DOI: 10.1073/pnas.1813204116 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ Abstract: Understanding molecular events during virus assembly and genome packaging is important for understanding viral life cycles, and the functioning of other protein–nucleic acid machines. The model system developed for the thermophilic bacteriophage P23-45 offers advantages over other systems. Cryo-EM reconstructions reveal modifications to a canonical capsid protein fold, resulting in capsids that are abnormally large for this virus class. Structural information on the portal protein, through which the genome is packaged, demonstrates that the capsid influences the portal’s conformation. This has implications for understanding how processes inside and outside the capsid can be coordinated.	Feb 2019
Krios I-Titan Krios I at Diamond	HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly Nikesh Patel , Simon J. White , Rebecca F. Thompson , Richard Bingham , Eva U. Weiß , Daniel P. Maskell , Adam Zlotnick , Eric C. Dykeman , Roman Tuma , Reidun Twarock , Neil A. Ranson , Peter G. Stockley Nature Microbiology 2 DOI: 10.1038/nmicrobiol.2017.98 Diamond Proposal Number(s): [11164] Show Abstract ▼ Abstract: Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein–RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.	Jun 2017
Krios I-Titan Krios I at Diamond	Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM Matthew G. Iadanza , Anna J. Higgins , Bob Schiffrin , Antonio N. Calabrese , David J. Brockwell , Alison E. Ashcroft , Sheena E. Radford , Neil A. Ranson Nature Communications 7 DOI: 10.1038/ncomms12865 Diamond Proposal Number(s): [12877, 13590] Open Access Show Abstract ▼ Abstract: The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA–E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.	Sep 2016
Krios I-Titan Krios I at Diamond	An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology Rebecca F. Thompson , Matt Walker , C. Alistair Siebert , Stephen P. Muench , Neil A. Ranson Methods 100, 3-15 DOI: 10.1016/j.ymeth.2016.02.017 Open Access Show Abstract ▼ Abstract: Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins greater than 150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a ’resolution revolution’, owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM.	Feb 2016

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