I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[12112, 11651]
Open Access
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)
|
Open Access
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
|
Diamond Proposal Number(s):
[21440]
Open Access
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
|
Diamond Proposal Number(s):
[11651]
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
|
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
Diamond Proposal Number(s):
[15616]
Open Access
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
|
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
Diamond Proposal Number(s):
[6638, 8659]
Open Access
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
|
Diamond Proposal Number(s):
[12346]
Open Access
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
|
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
Diamond Proposal Number(s):
[9306, 6638, 8659]
Open Access
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
|
Diamond Proposal Number(s):
[8659]
Open Access
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
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[5689]
Abstract: TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.
|
Sep 2014
|
|
