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58 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
I04-Macromolecular Crystallography	Macrocyclic peptide probes for immunomodulatory protein CD59: potent modulators of bacterial toxin activity and antibody-dependent cytotoxicity Edward W. Tate , Jasmine K. Bickel , Ammar. I. S. Ahmed , Aidan B. Pidd , Rhodri M. Morgan , Tom E. Mcallister , Sam M. Horrell , Emma C. Couves , Hemavathi Nagaraj , Edward J. Bartlett , Kamel El Omari , Akane Kawamura , Doryen Bubeck Angewandte Chemie International Edition DOI: 10.1002/anie.202422673 Diamond Proposal Number(s): [17221] Open Access Show Abstract ▼ Abstract: CD59 is an immunomodulatory cell surface receptor associated with human disease. Despite its importance in complement regulation and bacterial pathogenesis, CD59 remains a challenging therapeutic target. Research to date has focused on antibody or protein-based strategies. Here we present a new approach to target CD59 using macrocyclic peptides with low nanomolar affinity for CD59. Through X-ray crystallographic studies and structure-activity relationship studies we identify key interactions which are essential for binding and activity. We find that the macrocyclic peptide CP-06 adopts a beta-hairpin structure and binds CD59 through an intermolecular beta-sheet, mimicking protein-protein interactions of biologically relevant CD59 interaction partners. We create dimeric and lipidated macrocyclic peptide conjugates as enhanced cell-active CD59 inhibitors and show that these probes can be used to modulate both complement-mediated killing of human cells and lytic activity of bacterial virulence factors. Together, our data provide a starting point for future development of macrocyclic peptides to target CD59 activity in diverse cellular contexts.	Apr 2025
I23-Long wavelength MX	Light atoms identification and location by anomalous scattering Kamel El Omari , Ramona Duman , Christian Orr , Vitaliy Mykhaylyk , Armin Wagner 74th Annual Meeting Of The American Crystallographic Association DOI: 10.1063/4.0000427 Open Access Show Abstract ▼ Abstract: More than a third of all known proteins bind metal ions. Metal ions play key roles in a broad range of cellular processes, they are involved in protein structure stability and catalysis; with traditional examples of zinc fingers in transcription factors and iron in haemoglobin. Therefore, identifying metal ion-binding sites is important for understanding the biological functions of proteins and further helps in designing potent therapeutics. Experimental and computational methods have been developed to identify or predict metal ion ligand-binding residues. However, experimentally identifying and locating metal ions, such as calcium and potassium in protein structures can be challenging. The unique wavelength range of the macromolecular crystallography beamline I23 at Diamond Light Source allows identification and location of metal ions and lighter atoms of biological relevance (Ca, K, S, P and Cl) using X-ray anomalous scattering in crystal structure analysis. In a typical experiment, anomalous datasets are collected at two wavelengths, above and below the ion or element absorption edge, and then processed to calculate phased anomalous Fourier difference maps. The difference in anomalous peak heights between these two datasets allows the direct identification and visualisation of the ion in the protein structure. We successfully used this method in different projects to experimentally map ions in crystal structures and some examples will be discussed.	Mar 2025
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography Krios II-Titan Krios II at Diamond	Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide Audrey Le Bas , Bradley R. Clarke , Tanisha Teelucksingh , Micah Lee , Kamel El Omari , Andrew M. Giltrap , Stephen A. Mcmahon , Hui Liu , John H. Beale , Vitaliy Mykhaylyk , Ramona Duman , Neil G. Paterson , Philip N. Ward , Peter J. Harrison , Miriam Weckener , Els Pardon , Jan Steyaert , Huanting Liu , Andrew Quigley , Benjamin G. Davis , Armin Wagner , Chris Whitfield , James H. Naismith Open Biology 15 DOI: 10.1098/rsob.240310 Diamond Proposal Number(s): [33941] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.	Jan 2025
	Structure of the RNA-dependent RNA polymerase P2 from the cystovirus φ8 Merlyn Latimer-Smith , Paula S. Salgado , Ismay Forsyth , Eugene Makeyev , Minna M. Poranen , Dave I. Stuart , Jonathan M. Grimes , Kamel El Omari Scientific Reports 14 DOI: 10.1038/s41598-024-75213-7 Open Access Show Abstract ▼ Abstract: The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps.	Oct 2024
I23-Long wavelength MX	Utilizing anomalous signals for element identification in macromolecular crystallography Kamel El Omari , Ismay Forsyth , Ramona Duman , Christian M. Orr , Vitaliy Mykhaylyk , Erika J. Mancini , Armin Wagner Acta Crystallographica Section D Structural Biology 80 DOI: 10.1107/S2059798324008659 Open Access Show Abstract ▼ Abstract: AlphaFold2 has revolutionized structural biology by offering unparalleled accuracy in predicting protein structures. Traditional methods for determining protein structures, such as X-ray crystallography and cryo-electron microscopy, are often time-consuming and resource-intensive. AlphaFold2 provides models that are valuable for molecular replacement, aiding in model building and docking into electron density or potential maps. However, despite its capabilities, models from AlphaFold2 do not consistently match the accuracy of experimentally determined structures, need to be validated experimentally and currently miss some crucial information, such as post-translational modifications, ligands and bound ions. In this paper, the advantages are explored of collecting X-ray anomalous data to identify chemical elements, such as metal ions, which are key to understanding certain structures and functions of proteins. This is achieved through methods such as calculating anomalous difference Fourier maps or refining the imaginary component of the anomalous scattering factor f′′. Anomalous data can serve as a valuable complement to the information provided by AlphaFold2 models and this is particularly significant in elucidating the roles of metal ions.	Oct 2024
I03-Macromolecular Crystallography I23-Long wavelength MX	Structural insights into i-motif DNA structures in sequences from the insulin-linked polymorphic region Dilek Guneri , Effrosyni Alexandrou , Kamel El Omari , Zuzana Dvořáková , Rupesh V. Chikhale , Daniel T. S. Pike , Christopher A. Waudby , Christopher J. Morris , Shozeb Haider , Gary N. Parkinson , Zoë A. E. Waller Nature Communications 15 DOI: 10.1038/s41467-024-50553-0 Open Access Show Abstract ▼ Abstract: The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.	Aug 2024
I03-Macromolecular Crystallography	The effect of pH on the structure of Bluetongue virus VP5 Hanwen Zhang , Kamel El Omari , Geoff Sutton , David I. Stuart Journal Of General Virology 105 DOI: 10.1099/jgv.0.002018 Diamond Proposal Number(s): [8423, 10627] Open Access Show Abstract ▼ Abstract: The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.	Aug 2024
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography	Molecular mechanism of BMP signal control by Twisted gastrulation Tomas Malinauskas , Gareth Moore , Amalie F. Rudolf , Holly Eggington , Hayley L. Belnoue-Davis , Kamel El Omari , Samuel C. Griffiths , Rachel E. Woolley , Ramona Duman , Armin Wagner , Simon J. Leedham , Clair Baldock , Hilary L. Ashe , Christian Siebold Nature Communications 15 DOI: 10.1038/s41467-024-49065-8 Diamond Proposal Number(s): [28534, 14744, 19946] Open Access Show Abstract ▼ Abstract: Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.	Jun 2024
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography	Structure and function of Semaphorin-5A glycosaminoglycan interactions Gergely N. Nagy , Xiao-Feng Zhao , Richard Karlsson , Karen Wang , Ramona Duman , Karl Harlos , Kamel El Omari , Armin Wagner , Henrik Clausen , Rebecca L. Miller , Roman J. Giger , E. Yvonne Jones Nature Communications 15 DOI: 10.1038/s41467-024-46725-7 Diamond Proposal Number(s): [19946, 28534] Open Access Show Abstract ▼ Abstract: Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.	Mar 2024
B21-High Throughput SAXS I03-Macromolecular Crystallography I23-Long wavelength MX	Structural comparison of typical and atypical E2 pestivirus glycoproteins Hazel Aitkenhead , Christiane Riedel , Nathan Cowieson , Hans Tillmann Rümenapf , David I. Stuart , Kamel El Omari Structure 10 DOI: 10.1016/j.str.2023.12.003 Open Access Show Abstract ▼ Abstract: Pestiviruses, within the family Flaviviridae, are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed β-hairpin reported to be involved in host receptor binding. Functional analysis of this β-hairpin in the context of BVDV revealed its ability to rescue viral infectivity.	Jan 2024

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