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

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS I03-Macromolecular Crystallography	Non-canonical Staphylococcus aureus pathogenicity island repression Laura Miguel-Romero , Mohammed Alqasmi , Julio Bacarizo , Jason A. Tan , Richard J. Cogdell , John Chen , Olwyn Byron , Gail E. Christie , Alberto Marina , José R. Penadés Nucleic Acids Research 2 DOI: 10.1093/nar/gkac855 Diamond Proposal Number(s): [16258] Open Access Show Abstract ▼ Abstract: Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.	Oct 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	High-resolution structure of the alcohol dehydrogenase domain of the bifunctional bacterial enzyme AdhE Liyana Azmi , Eilis C. Bragginton , Ian T. Cadby , Olwyn Byron , Andrew Roe , Andrew L. Lovering , Mads Gabrielsen Acta Crystallographica Section F Structural Biology Communications 76, 414 - 421 DOI: 10.1107/S2053230X20010237 Diamond Proposal Number(s): [12112, 11651] Open Access Show Abstract ▼ Abstract: The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.	Sep 2020
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for centromere maintenance by Drosophila CENP ‐A chaperone CAL 1 Bethan Medina-Pritchard , Vasiliki Lazou , Juan Zou , Olwyn Byron , Maria A. Abad , Juri Rappsilber , Patrick Heun , A. Arockia Jeyaprakash The Embo Journal 7 DOI: 10.15252/embj.2019103234 Open Access Show Abstract ▼ Abstract: Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP‐A‐containing nucleosomes. To preserve centromere identity, CENP‐A must be escorted to centromeres by a CENP‐A‐specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP‐A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X‐ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP‐A histone chaperone, binds both CENP‐A and the centromere receptor CENP‐C without the requirement for the Mis18 complex. While an N‐terminal CAL1 fragment wraps around CENP‐A/H4 through multiple physical contacts, a C‐terminal CAL1 fragment directly binds a CENP‐C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP‐A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP‐C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP‐C dimer. Overall, by demonstrating how CAL1 binds CENP‐A/H4 and CENP‐C, we provide key insights into the minimalistic principles underlying centromere maintenance.	Mar 2020
B21-High Throughput SAXS	Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity Gijeong Kim , Liyana Azmi , Seongmin Jang , Taeyang Jung , Hans Hebert , Andrew J. Roe , Olwyn Byron , Ji-Joon Song Nature Communications 10 DOI: 10.1038/s41467-019-12427-8 Diamond Proposal Number(s): [21440] Open Access Show Abstract ▼ Abstract: Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.	Oct 2019
B21-High Throughput SAXS	Calicivirus VP2 forms a portal-like assembly following receptor engagement Michaela J. Conley , Marion Mcelwee , Liyana Azmi , Mads Gabrielsen , Olwyn Byron , Ian G. Goodfellow , David Bhella Nature 565, 377 - 381 DOI: 10.1038/s41586-018-0852-1 Diamond Proposal Number(s): [11651] Show Abstract ▼ Abstract: To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.	Jan 2019
B21-High Throughput SAXS	Merging in-solution x-ray and neutron scattering data allows fine structural analysis of membrane–protein detergent complexes Gaetan Dias Mirandela , Giulia Tamburrino , Miloš T. Ivanović , Felix M. Strnad , Olwyn Byron , Tim Rasmussen , Paul A. Hoskisson , Jochen S. Hub , Ulrich Zachariae , Frank Gabel , Arnaud Javelle The Journal Of Physical Chemistry Letters DOI: 10.1021/acs.jpclett.8b01598 Diamond Proposal Number(s): [15616] Open Access Show Abstract ▼ Abstract: In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein–detergent complexes from unlabeled samples than SAXS or SANS alone.	Jul 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure of the bacterial plant-ferredoxin receptor FusA Rhys Grinter , Inokentijs Josts , Khedidja Mosbahi , Aleksander W. Roszak , Richard J. Cogdell , Alexandre M. J. J. Bonvin , Joel J. Milner , Sharon M. Kelly , Olwyn Byron , Brian O. Smith , Daniel Walker Nature Communications 7 DOI: 10.1038/ncomms13308 Diamond Proposal Number(s): [6638, 8659] Open Access Show Abstract ▼ Abstract: Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens. DOI: 10.1038/ncomms13308 OPEN 1	Oct 2016
B21-High Throughput SAXS I02-Macromolecular Crystallography Data acquisition Diagnostics Health Physics	Structural and biophysical analysis of nuclease protein antibiotics Alexander Klein , Justyna Wojdyla , Amar Joshi , Inokentijs Josts , L. C. Mccaughey , N. Housden , R. Kaminska , Olwyn Byron , D. Walker , C. Kleanthous Biochemical Journal DOI: 10.1042/BCJ20160544 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa , known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity immunity protein, Im9. In contrast to Im3, which when bound to the ribonuclease (rRNase) domain of the homologous colicin ColE3 makes contact with the translocation (T-) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small angle X-ray scattering (SAXS) data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.	Jul 2016
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structures of the ultra-high-affinity protein–protein complexes of pyocins S2 and AP41 and their cognate immunity proteins from Pseudomonas aeruginosa Amar Joshi , Rhys Grinter , Inokentijs Josts , Sabrina Chen , Justyna Wojdyla , Edward D. Lowe , Renata Kaminska , Connor Sharp , Laura Mccaughey , Aleksander Roszak , Richard J. Cogdell , Olwyn Byron , Daniel Walker , Colin Kleanthous Journal Of Molecular Biology 427 (17), 2852 - 2866 DOI: 10.1016/j.jmb.2015.07.014 PMID: 26215615 Diamond Proposal Number(s): [9306, 6638, 8659] Open Access Show Abstract ▼ Abstract: How ultra-high-affinity protein protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase Im subfamilies and are important in extending our understanding of protein protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase Immunity pairs that appear to underpin the split of this family into two distinct groups. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).	Aug 2015
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure of protease-cleaved Escherichia coli alpha-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment Cameron Fyfe , Rhys Grinter , Inokentijs Josts , Khedidja Mosbahi , Aleksander Roszak , Richard J. Cogdell , Daniel M. Wall , Richard J. S. Burchmore , Olwyn Byron , Daniel Walker Acta Crystallographica Section D Biological Crystallography 71 (7), 1478 - 1486 DOI: 10.1107/S1399004715008548 PMID: 26143919 Diamond Proposal Number(s): [8659] Open Access Show Abstract ▼ Abstract: Bacterial alpha-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli alpha-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli alpha-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli alpha-2-macroglobulin and human alpha-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.	Jul 2015

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