Krios IV-Titan Krios IV at Diamond
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Alister
Burt
,
C. Keith
Cassidy
,
Peter
Ames
,
Maria
Bacia-verloop
,
Megghane
Baulard
,
Karine
Huard
,
Zaida
Luthey-schulten
,
Ambroise
Desfosses
,
Phillip J.
Stansfeld
,
William
Margolin
,
John S.
Parkinson
,
Irina
Gutsche
Diamond Proposal Number(s):
[18112]
Open Access
Abstract: Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.
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Feb 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tao
Ni
,
Fang
Jiao
,
Xiulian
Yu
,
Saša
Aden
,
Lucy
Ginger
,
Sophie I.
Williams
,
Fangfang
Bai
,
Vojtech
Prazak
,
Dimple
Karia
,
Phillip
Stansfeld
,
Peijun
Zhang
,
George
Munson
,
Gregor
Anderluh
,
Simon
Scheuring
,
Robert J. C.
Gilbert
Abstract: Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
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Jan 2020
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I24-Microfocus Macromolecular Crystallography
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Simon R.
Bushell
,
Ashley C. W.
Pike
,
Maria E.
Falzone
,
Nils J. G.
Rorsman
,
Chau M.
Ta
,
Robin A.
Corey
,
Thomas D.
Newport
,
John C.
Christianson
,
Lara F.
Scofano
,
Chitra
Shintre
,
Annamaria
Tessitore
,
Amy
Chu
,
Qinrui
Wang
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
James D.
Love
,
Nicola A.
Burgess-brown
,
Rebecca
Sitsapesan
,
Phillip J.
Stansfeld
,
Juha T.
Huiskonen
,
Paolo
Tammaro
,
Alessio
Accardi
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[10619, 15433]
Open Access
Abstract: Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
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Sep 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.
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Mar 2018
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I24-Microfocus Macromolecular Crystallography
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Meriem
El Ghachi
,
Nicole
Howe
,
Chia-ying
Huang
,
Vincent
Olieric
,
Rangana
Warshamanage
,
Thierry
Touzé
,
Dietmar
Weichert
,
Phillip J.
Stansfeld
,
Meitian
Wang
,
Fred
Kerff
,
Martin
Caffrey
Diamond Proposal Number(s):
[12710]
Open Access
Abstract: As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.
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Mar 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) and Knob-associated Histidine-rich Protein (KAHRP) are directly linked to malaria pathology. PfEMP1 and KAHRP cluster on protrusions (knobs) on the P. falciparum-infected erythrocyte surface and enable pathogenic cytoadherence of infected erythrocytes to the host microvasculature, leading to restricted blood flow, oxygen deprivation and damage of tissues. Here we characterize the interactions of PfEMP1 and KAHRP with host erythrocyte spectrin using biophysical, structural and computational approaches. These interactions assist knob formation and, thus, promote cytoadherence. We show that the folded core of the PfEMP1 cytosolic domain interacts broadly with erythrocyte spectrin but shows weak, residue-specific preference for domain 17 of α spectrin, which is proximal to the erythrocyte cytoskeletal junction. In contrast, a protein sequence repeat region in KAHRP preferentially associates with domains 10–14 of β spectrin, proximal to the spectrin–ankyrin complex. Structural models of PfEMP1 and KAHRP with spectrin combined with previous microscopy and protein interaction data suggest a model for knob architecture.
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Aug 2017
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I24-Microfocus Macromolecular Crystallography
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Maciej
Wiktor
,
Dietmar
Weichert
,
Nicole
Howe
,
Chia-ying
Huang
,
Vincent
Olieric
,
Coilín
Boland
,
Jonathan
Bailey
,
Lutz
Vogeley
,
Phillip J.
Stansfeld
,
Nienke
Buddelmeijer
,
Meitian
Wang
,
Martin
Caffrey
Diamond Proposal Number(s):
[11890, 12710]
Open Access
Abstract: Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.
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Jul 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Yinghong
Gu
,
Huanyu
Li
,
Haohao
Dong
,
Yi
Zeng
,
Zhengyu
Zhang
,
Neil G.
Paterson
,
Phillip J.
Stansfeld
,
Zhongshan
Wang
,
Yizheng
Zhang
,
Wenjian
Wang
,
Changjiang
Dong
Diamond Proposal Number(s):
[9475]
Abstract: All Gram-negative bacteria, mitochondria and chloroplasts have outer membrane proteins (OMPs) that perform many fundamental biological processes. The OMPs in Gram-negative bacteria are inserted and folded into the outer membrane by the β-barrel assembly machinery (BAM). The mechanism involved is poorly understood, owing to the absence of a structure of the entire BAM complex. Here we report two crystal structures of the Escherichia coli BAM complex in two distinct states: an inward-open state and a lateral-open state. Our structures reveal that the five polypeptide transport-associated domains of BamA form a ring architecture with four associated lipoproteins, BamB–BamE, in the periplasm. Our structural, functional studies and molecular dynamics simulations indicate that these subunits rotate with respect to the integral membrane β-barrel of BamA to induce movement of the β-strands of the barrel and promote insertion of the nascent OMP.
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Feb 2016
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I24-Microfocus Macromolecular Crystallography
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Abstract: With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.
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Feb 2016
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I24-Microfocus Macromolecular Crystallography
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Dianfan
Li
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aaron
Keogh
,
Lutz
Vogeley
,
Nicole
Howe
,
Joseph
Lyons
,
David
Aragao
,
Petra
Fromme
,
Raimund
Fromme
,
Shibom
Basu
,
Ingo
Grotjohann
,
Christopher
Kupitz
,
Kimberley
Rendek
,
Uwe
Weierstall
,
Nadia A.
Zatsepin
,
Vadim
Cherezov
,
Wei
Liu
,
Sateesh
Bandaru
,
Niall J.
English
,
Cornelius
Gati
,
Anton
Barty
,
Oleksandr
Yefanov
,
Henry N.
Chapman
,
Kay
Diederichs
,
Marc
Messerschmidt
,
Sébastien
Boutet
,
Garth J.
Williams
,
M.
Marvin Seibert
,
Martin
Caffrey
Open Access
Abstract: Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
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Dec 2015
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