I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Liuyi
Hu
,
Alan
Wainman
,
Antonina
Andreeva
,
Muladili
Apizi
,
Ines
Alvarez-Rodrigo
,
Siu-Shing
Wong
,
Saroj
Saurya
,
Devon
Sheppard
,
Matthew
Cottee
,
Steven
Johnson
,
Susan M.
Lea
,
Jordan W.
Raff
,
Mark
Van Breugel
,
Zhe
Feng
Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Centrosomes form when centrioles assemble pericentriolar material (PCM) around themselves. Spd-2/CEP192 proteins, defined by a conserved “Spd-2 domain” (SP2D) comprising two closely spaced AspM-Spd-2-Hydin (ASH) domains, play a critical role in centrosome assembly. Here, we show that the SP2D does not target Drosophila Spd-2 to centrosomes but rather promotes PCM scaffold assembly. Crystal structures of the human and honeybee SP2D reveal an unusual “extended cradle” structure mediated by a conserved interaction interface between the two ASH domains. Mutations predicted to perturb this interface, including a human mutation associated with male infertility and Mosaic Variegated Aneuploidy, disrupt PCM scaffold assembly in flies. The SP2D is monomeric in solution, but the Drosophila SP2D can form higher-order oligomers upon phosphorylation by PLK1 (Polo-like kinase 1). Crystal-packing interactions and AlphaFold predictions suggest how SP2Ds might self-assemble, and mutations associated with one such potential dimerization interface markedly perturb SP2D oligomerization in vitro and PCM scaffold assembly in vivo.
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Mar 2025
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Zhihan
Bo
,
Thomas
Rowntree
,
Steven
Johnson
,
Hilman
Nurmahdi
,
Richard J.
Suckling
,
Johan
Hill
,
Boguslawa
Korona
,
Philip C.
Weisshuhn
,
Devon
Sheppard
,
Yao
Meng
,
Shaoyan
Liang
,
Edward D.
Lowe
,
Susan M.
Lea
,
Christina
Redfield
,
Penny A.
Handford
Diamond Proposal Number(s):
[31353, 7495, 12346]
Open Access
Abstract: The Notch receptor is activated by the Delta/Serrate/Lag-2 (DSL) family of ligands. The organization of the extracellular signaling complex is unknown, although structures of Notch/ligand complexes comprising the ligand-binding region (LBR), and negative regulatory region (NRR) region, have been solved. Here, we investigate the human Notch-1 epidermal growth factor-like (EGF) 20-27 region, located between the LBR and NRR, and incorporating the Abruptex (Ax) region, associated with distinctive Drosophila phenotypes. Our analyses, using crystallography, NMR and small angle X-ray scattering (SAXS), support a rigid, elongated organization for EGF20-27 with the EGF20-21 linkage showing Ca2+-dependent flexibility. In functional assays, Notch-1 variants containing Ax substitutions result in reduced ligand-dependent trans-activation. When cis-JAG1 was expressed, Notch activity differences between WT and Ca2+-binding Ax variants were less marked than seen in the trans-activation assays alone, consistent with disruption of cis-inhibition. These data indicate the importance of Ca2+-stabilized structure and suggest the balance of cis- and trans-interactions explains the effects of Drosophila Ax mutations.
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Nov 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346, 18069]
Open Access
Abstract: Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.
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Jan 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Shigella sonnei is a major cause of bacillary dysentery, and of increasing concern due to the spread of multi-drug resistance.S. sonneiharbours pINV, a ∼ 210 kb plasmid that encodes a Type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously inS. sonneiat high frequency, hampering studies on and vaccine development against this important pathogen. Here we investigated the molecular basis for the emergence of avirulence inS. sonnei, and show that avirulence mainly results from pINV loss, consistent with previous findings. Ancestral deletions have led to the loss fromS. sonneipINV of two toxin:antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV inShigella flexneri. We show that introduction of these TA systems intoS. sonneipINV reduced but did not eliminate pINV loss, while single amino acid polymorphisms found in theS. sonneiVapBC TA system compared withS. flexneriVapBC also contribute to pINV loss. Avirulence also results from deletions of T3SS-associated genes on pINV through recombination between insertion sequences (ISs) on the plasmid; these events differ from those observed inS. flexneridue to the different distribution and repertoire of ISs. Our findings demonstrate that TA systems and ISs influence plasmid dynamics and loss inS. sonnei, and could be exploited for the design and evaluation of vaccines.
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Jan 2022
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
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.
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Aug 2021
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Open Access
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.
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Feb 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
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.
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Feb 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
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.
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Dec 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
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.
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Mar 2018
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B22-Multimode InfraRed imaging And Microspectroscopy
I11-High Resolution Powder Diffraction
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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
Diamond Proposal Number(s):
[14341, 14938]
Open Access
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).
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Oct 2017
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