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

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling Torcato Martins , Yao Meng , Boguslawa Korona , Richard Suckling , Steven Johnson , Penny A Handford , Susan M. Lea , Sarah J Bray Embo Reports 8 DOI: 10.15252/embr.202152729 Open Access Show Abstract ▼ Abstract: Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called β1-2 loop that is involved in phospholipid binding. Mutations in the β1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand–receptor interactions.	Aug 2021
	Hydrophilic and hydrophobic interactions in concentrated aqueous imidazole solutions: a neutron diffraction and total X-ray scattering study Laila H. Al-Madhagi , Sam Callear , Sven L. M. Schroeder Physical Chemistry Chemical Physics 390 DOI: 10.1039/C9CP05993H Open Access Show Abstract ▼ Abstract: The intermolecular interactions in concentrated (5 M) aqueous imidazole solutions have been investigated by combining neutron diffraction with isotopic substitution, total X-ray scattering and empirical potential structure refinement (EPSR) simulations using a box containing 5530 water and 500 imidazole molecules. The structural model with the best fit was used to generate radial distribution functions and spatial density functions. The local volume surrounding imidazole molecules is dominated by water, due to strong hydrogen-bonding between the nitrogen moieties of imidazole and water molecules; within a radius of 6.4 Å from the central imidazole molecule there are, on average, 17 water and only 3 imidazole molecules. Even though imidazole interacts with water it appears to disrupt hydrogen bonding in the surrounding water network only minimally. Hydrogen-bonding between imidazole molecules is negligible. The most probable positions of imidazole nearest-neighbours are above and below the plane of the aromatic ring. At low distances (up to ∼3.5–3.8 Å) these nearest neighbours were found to prefer parallel orientation of the molecular planes, indicating hydrophobic (π–π) stacking. At longer distances (up to ∼5 Å), imidazole neighbours assume both parallel and edge-to-face orientations. Overall, hydrated imidazole molecules are the most probable structural motif in aqueous solutions, with very few direct imidazole–imidazole interactions.	Feb 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold Martin P. Reichhardt , Vuokko Loimaranta , Susan M. Lea , Steven Johnson Life Science Alliance 3 DOI: 10.26508/lsa.201900502 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.	Feb 2020
I03-Macromolecular Crystallography	An inhibitor of complement C5 provides structural insights into activation Martin P. Reichhardt , Steven Johnson , Terence Tang , Thomas Morgan , Nchimunya Tebeka , Niko Popitsch , Justin C. Deme , Matthijs M. Jore , Susan Lea Proceedings Of The National Academy Of Sciences 117 DOI: 10.1073/pnas.1909973116 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5–CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4–CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.	Dec 2019
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structure-based design of chimeric antigens for multivalent protein vaccines S. Hollingshead , I. Jongerius , R. M. Exley , S. Johnson , S. Lea , C. M. Tang Nature Communications 9 DOI: 10.1038/s41467-018-03146-7 Open Access Show Abstract ▼ Abstract: There is an urgent need to develop vaccines against pathogenic bacteria. However, this is often hindered by antigenic diversity and difficulties encountered manufacturing membrane proteins. Here we show how to use structure-based design to develop chimeric antigens (ChAs) for subunit vaccines. ChAs are generated against serogroup B Neisseria meningitidis (MenB), the predominant cause of meningococcal disease in wealthy countries. MenB ChAs exploit factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and the PorA VR2 epitope adopts an immunogenic conformation. In mice, immunisation with ChAs generates fHbp and PorA antibodies that recognise the antigens expressed by clinical MenB isolates; these antibody responses correlate with protection against meningococcal disease. Application of ChAs is therefore a potentially powerful approach to develop multivalent subunit vaccines, which can be tailored to circumvent pathogen diversity.	Mar 2018
B22-Multimode InfraRed imaging And Microspectroscopy I11-High Resolution Powder Diffraction	Confinement of iodine molecules into triple-helical chains within robust metal–organic frameworks Xinran Zhang , Ivan Da Silva , Harry G. W. Godfrey , Samantha K. Callear , Sergey A. Sapchenko , Yongqiang Cheng , Inigo J. Vitorica-yrezabal , Mark D. Frogley , Gianfelice Cinque , Chiu C. Tang , Carlotta Giacobbe , Catherine Dejoie , Svemir Rudic , Anibal J. Ramirez-cuesta , Melissa A. Denecke , Sihai Yang , Martin Schroeder Journal Of The American Chemical Society DOI: 10.1021/jacs.7b08748 Diamond Proposal Number(s): [14341, 14938] Show Abstract ▼ Abstract: During the nuclear waste disposal process, radioactive iodine in fission product can be released. The widespread implementation of sustainable nuclear energy thus requires the development of efficient iodine stores that have simultaneously high capacity, stability and more importantly, storage density (and hence minimised system volume). Here, we report high I2 adsorption in a series of robust porous metal-organic materials, MFM-300(M) (M = Al, Sc, Fe, In). MFM-300(Sc) exhibits fully reversible I2 uptake of 1.54 g g-1 and its structure remains completely unperturbed upon inclusion/removal of I2. Direct observation and quantification of the adsorption, binding domains and dynamics of guest I2 molecules within these hosts have been achieved using XPS, TGA-MS, high resolution synchrotron X-ray diffraction, pair distribution function analysis, Raman, terahertz and neutron spectroscopy, coupled with density functional theory modelling. These complimentary techniques reveal a comprehensive understanding on the host-I2 and I2-I2 binding interaction at a molecular level. The initial binding site of I2 in MFM-300(Sc), I2I, is located near the bridging hydroxyl group of the [ScO4(OH)2] moiety [I2I···H–O = 2.263(9) Å] with an occupancy of 0.268. I2II is located interstitially between two phenyl rings of neighbouring ligand molecules [I2II···phenyl ring = 3.378(9) and 4.228(5) Å]. I2II is 4.565(2) Å from the hydroxyl group with an occupancy of 0.208. Significantly, at high I2 loading an unprecedented self-aggregation of I2 molecules into triple-helical chains within the confined nano-voids has been observed at crystallographic resolution, leading to a highly efficient packing of I2 molecules with an exceptional I2 storage density of 3.08 g cm-3 in MFM-300(Sc).	Oct 2017
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural Basis for Mitotic Centrosome Assembly in Flies Zhe Feng , Anna Caballe , Alan Wainman , Steven Johnson , Andreas F. M. Haensele , Matthew A. Cottee , Paul T. Conduit , Susan M. Lea , Jordan W. Raff Cell 169, 1078 - 1089.e13 DOI: 10.1016/j.cell.2017.05.030 Diamond Proposal Number(s): [9306, 12346] Open Access Show Abstract ▼ Abstract: In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.	Jun 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands Richard J. Suckling , Boguslawa Korona , Pat Whiteman , Chandramouli Chillakuri , Laurie Holt , Penny A. Handford , Susan M. Lea The Embo Journal DOI: 10.15252/embj.201796632 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.	Jun 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	A key centriole assembly interaction interface between human Plk4 and STIL appears to not be conserved in flies Matthew A. Cottee , Steven Johnson , Jordan W. Raff , Susan M. Lea Biology Open DOI: 10.1242/bio.024661 Diamond Proposal Number(s): [9306] Open Access Show Abstract ▼ Abstract: A small number of proteins form a conserved pathway of centriole duplication. In humans and flies, the binding of Plk4/Sak to STIL/Ana2 initiates daughter centriole assembly. In humans, this interaction is mediated by an interaction between the Polo-Box-3 (PB3) domain of Plk4 and the coiled-coil domain of STIL (HsCCD). We showed previously that the Drosophila Ana2 coiled-coil domain (DmCCD) is essential for centriole assembly, but it forms a tight parallel tetramer in vitro that likely precludes an interaction with PB3. Here we show that the isolated HsCCD and HsPB3 domains form a mixture of homo-multimers in vitro, but these readily dissociate when mixed to form the previously described 1:1 HsCCD:HsPB3 complex. In contrast, although Drosophila PB3 (DmPB3) adopts a canonical polo-box fold, it does not detectably interact with DmCCD in vitro. Thus, surprisingly, a key centriole assembly interaction interface appears to differ between humans and flies.	Feb 2017
I02-Macromolecular Crystallography Data acquisition Detectors Diagnostics Health Physics	A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding Jamie Berry , Yingqi Xu , Philip n. Ward , Susan Lea , Steve Matthews , Vladimir Pelicic Structure 24, 926 - 934 DOI: 10.1016/j.str.2016.04.001 Show Abstract ▼ Abstract: DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.	Jun 2016

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