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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I11-High Resolution Powder Diffraction
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Open Access
Abstract: Stimuli-responsive behaviors of flexible metal–organic frameworks (MOFs) make these materials promising in a wide variety of applications such as gas separation, drug delivery, and molecular sensing. Considerable efforts have been made over the last decade to understand the structural changes of flexible MOFs in response to external stimuli. Uniform pore deformation has been used as the general description. However, recent advances in synthesizing MOFs with non-uniform porous structures, i.e. with multiple types of pores which vary in size, shape, and environment, challenge the adequacy of this description. Here, we demonstrate that the CO2-adsorption-stimulated structural change of a flexible MOF, ZIF-7, is induced by CO2 migration in its non-uniform porous structure rather than by the proactive opening of one type of its guest-hosting pores. Structural dynamics induced by guest migration in non-uniform porous structures is rare among the enormous number of MOFs discovered and detailed characterization is very limited in the literature. The concept presented in this work provides new insights into MOF flexibility.
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Mar 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]
Open Access
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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Abstract: Ubiquitin specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15’s catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ~10Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active site loops are flexible, resulting in a largely open ubiquitin tail binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1’ region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin and linear diubiquitin binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1’ binding site. Our results reveal first insights into USP15’s catalytic domain structure, conformational changes, differences between paralogs, and small molecule interactions and establish a framework for cellular probe and inhibitor development.
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Sep 2018
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B18-Core EXAFS
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Grazia
Malta
,
Simon A.
Kondrat
,
Simon J.
Freakley
,
Catherine
Davies
,
Simon
Dawson
,
Xi
Liu
,
Li
Lu
,
Krzysztof
Dymkowski
,
Felix
Fernandez-Alonso
,
Sanghamitra
Mukhopadhyay
,
Emma Kate
Gibson
,
Peter P.
Wells
,
Stewart F.
Parker
,
Christopher J.
Kiely
,
Graham J.
Hutchings
Diamond Proposal Number(s):
[10306, 11398, 15214]
Open Access
Abstract: Single-site Au species supported on carbon have been shown to be the active sites for acetylene hydrochlorination. The evolution of these single-site species has been monitored by Au L3 X-ray Absorption Spectroscopy (XAS). Alternating between a standard reaction mixture of HCl/C2H2 and the single reactants, has provided insights into the reaction mechanism and catalyst deactivation processes. We demonstrate that oxidative addition of HCl across an Au(I) chloride species requires concerted addition with C2H2, in accordance with both the XAS measurements of Au oxidation state and the reaction kinetics being 1st order with respect to each reactant. The addition of excess C2H2 changes the Au speciation and results in the formation of oligomeric acetylene species which were detected by inelastic neutron scattering. Catalyst deactivation at extended reaction times can be correlated with the formation of metallic Au particles. The presence of this Au(0) species generated during the sequential gas experiments or after prolonged reaction times, results in the analysis of the normalised near edge white line intensity of the Au L3 X-ray absorption spectrum alone becoming an unsuitable guide for identifying the active Au species, affecting the strong correlation between normalized white line height and VCM productivity usually observed in the active catalyst. Thus, a combination of scanning transmission electron microscopy and detailed modelling of whole XAS spectrum was required to distinguish active Au(I) and Au(III) species from the spectator Au(0) component.
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Jul 2018
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[10215]
Abstract: We show clear experimental evidence of cooperative terahertz (THz) dynamics observed below 3 THz (∼100 cm−1), for a low-symmetry Zr-based metal-organic framework structure, termed MIL–140A [ZrO(O2C−C6H4−CO2)]. Utilizing a combination of high-resolution inelastic neutron scattering and synchrotron radiation far-infrared spectroscopy, we measured low-energy vibrations originating from the hindered rotations of organic linkers, whose energy barriers and detailed dynamics have been elucidated via ab initio density functional theory calculations. The complex pore architecture caused by the THz rotations has been characterized. We discovered an array of soft modes with trampolinelike motions, which could potentially be the source of anomalous mechanical phenomena such as negative thermal expansion. Our results demonstrate coordinated shear dynamics (2.47 THz), a mechanism which we have shown to destabilize the framework structure, in the exact crystallographic direction of the minimum shear modulus (Gmin).
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Jun 2017
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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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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Y. Y.
Dong
,
A.
Pike
,
A.
Mackenzie
,
C.
Mcclenaghan
,
P.
Aryal
,
L.
Dong
,
A.
Quigley
,
M.
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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