Krios I-Titan Krios I at Diamond
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Qinrui
Wang
,
Robin A.
Corey
,
George
Hedger
,
Prafulla
Aryal
,
Mariana
Grieben
,
Chady
Nasrallah
,
Agnese
Baronina
,
Ashley C. W.
Pike
,
Jiye
Shi
,
Elisabeth P.
Carpenter
,
Mark S. P.
Sansom
Diamond Proposal Number(s):
[14856]
Open Access
Abstract: Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
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Dec 2019
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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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I24-Microfocus Macromolecular Crystallography
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Marcus
Schewe
,
Han
Sun
,
Ümit
Mert
,
Alexandra
Mackenzie
,
Ashley C. W.
Pike
,
Friederike
Schulz
,
Cristina
Constantin
,
Kirsty S.
Vowinkel
,
Linus J.
Conrad
,
Aytug K.
Kiper
,
Wendy
Gonzalez
,
Marianne
Musinszki
,
Marie
Tegtmeier
,
David C.
Pryde
,
Hassane
Belabed
,
Marc
Nazare
,
Bert L.
De Groot
,
Niels
Decher
,
Bernd
Fakler
,
Elisabeth P.
Carpenter
,
Stephen J.
Tucker
,
Thomas
Baukrowitz
Diamond Proposal Number(s):
[8421]
Abstract: Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.
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Feb 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Yin Yao
Dong
,
Hua
Wang
,
Ashley C. W.
Pike
,
Stephen A.
Cochrane
,
Sadra
Hamedzadeh
,
Filip J.
Wyszyński
,
Simon R.
Bushell
,
Sylvain F.
Royer
,
David A.
Widdick
,
Andaleeb
Sajid
,
Helena I.
Boshoff
,
Yumi
Park
,
Ricardo
Lucas
,
Wei-min
Liu
,
Seung Seo
Lee
,
Takuya
Machida
,
Leanne
Minall
,
Shahid
Mehmood
,
Katsiaryna
Belaya
,
Wei-wei
Liu
,
Amy
Chu
,
Leela
Shrestha
,
Shubhashish M. M.
Mukhopadhyay
,
Claire
Strain-damerell
,
Rod
Chalk
,
Nicola A.
Burgess-brown
,
Mervyn J.
Bibb
,
Clifton E.
Barry
,
Carol V.
Robinson
,
David
Beeson
,
Benjamin G.
Davis
,
Elizabeth P.
Carpenter
Diamond Proposal Number(s):
[10619, 15433, 19301]
Abstract: Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Mariana
Grieben
,
Ashley C. W.
Pike
,
Chitra A.
Shintre
,
Elisa
Venturi
,
Sam
El-ajouz
,
Annamaria
Tessitore
,
Leela
Shrestha
,
Shubhashish
Mukhopadhyay
,
Pravin
Mahajan
,
Rod
Chalk
,
Nicola A
Burgess-brown
,
Rebecca
Sitsapesan
,
Juha T.
Huiskonen
,
Elisabeth P.
Carpenter
Diamond Proposal Number(s):
[10619]
Abstract: Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
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Dec 2016
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Open Access
Abstract: Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.
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Mar 2016
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I04-Macromolecular Crystallography
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Ashley
Pike
,
Shivasankari
Gomathinayagam
,
Paolo
Swuec
,
Matteo
Berti
,
Ying
Zhang
,
Christina
Schnecke
,
Francesca
Marino
,
Frank
Von Delft
,
Ludovic
Renault
,
Alessandro
Costa
,
Opher
Gileadi
,
Alessandro
Vindigni
Diamond Proposal Number(s):
[442]
Open Access
Abstract: RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.
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Apr 2015
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Y. Y.
Dong
,
Ashley
Pike
,
Alexandra
Mackenzie
,
C.
Mcclenaghan
,
P.
Aryal
,
L.
Dong
,
Andrew
Quigley
,
Mariana
Grieben
,
S.
Goubin
,
S.
Mukhopadhyay
,
G. F.
Ruda
,
M. V.
Clausen
,
L.
Cao
,
P. E.
Brennan
,
N. A.
Burgess-brown
,
M. S. P.
Sansom
,
S. J.
Tucker
,
E. P.
Carpenter
Diamond Proposal Number(s):
[8421, 10619]
Abstract: TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
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Mar 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Modern synchrotron beamlines offer instrumentation of unprecedented quality, which in turn encourages increasingly marginal experiments, and for these, as much as ever, the ultimate success of data collection depends on the experience, but especially the care, of the experimenter. A representative set of difficult cases has been encountered at the Structural Genomics Consortium, a worldwide structural genomics initiative of which the Oxford site currently deposits three novel human structures per month. Achieving this target relies heavily on frequent visits to the Diamond Light Source, and the variety of crystal systems still demand customized data collection, diligent checks and careful planning of each experiment. Here, an overview is presented of the techniques and procedures that have been refined over the years and that are considered synchrotron best practice.
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Jul 2013
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I24-Microfocus Macromolecular Crystallography
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Chitra A.
Shintre
,
Ashley C. W.
Pike
,
Quihong
Li
,
Jung-in
Kim
,
Alistair J.
Barr
,
Solenne
Goubin
,
Leela
Shrestha
,
Jingjie
Yang
,
Georgina
Berridge
,
Jonathan
Ross
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aled M.
Edwards
,
Chas
Bountra
,
Brian D.
Marsden
,
Frank
Von Delft
,
Alex N.
Bullock
,
Opher
Gileadi
,
Nicola A.
Burgess-brown
,
Elisabeth P.
Carpenter
Abstract: ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.
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May 2013
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