Krios I-Titan Krios I at Diamond
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Dmitry
Ghilarov
,
Satomi
Inaba-Inoue
,
Piotr
Stepien
,
Feng
Qu
,
Elizabeth
Michalczyk
,
Zuzanna
Pakosz
,
Norimichi
Nomura
,
Satoshi
Ogasawara
,
Graham Charles
Walker
,
Sylvie
Rebuffat
,
So
Iwata
,
Jonathan
Gardiner Heddle
,
Konstantinos
Beis
Diamond Proposal Number(s):
[18659]
Open Access
Abstract: Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.
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Sep 2021
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Tetsuo
Yamashita
,
Daniel Ken
Inaoka
,
Tomoo
Shiba
,
Takumi
Oohashi
,
So
Iwata
,
Takao
Yagi
,
Hiroaki
Kosaka
,
Hideto
Miyoshi
,
Shigeharu
Harada
,
Kiyoshi
Kita
,
Katsuya
Hirano
Open Access
Abstract: Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.
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Feb 2018
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B22-Multimode InfraRed imaging And Microspectroscopy
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Christopher D. M.
Hutchison
,
Violeta
Cordon-Preciado
,
Rhodri M. L.
Morgan
,
Takanori
Nakane
,
Josie
Ferreira
,
Gabriel
Dorlhiac
,
Alvaro
Sanchez-Gonzalez
,
Allan S.
Johnson
,
Ann
Fitzpatrick
,
Clyde
Fare
,
Jon
Marangos
,
Chun Hong
Yoon
,
Mark S.
Hunter
,
Daniel P.
Deponte
,
Sébastien
Boutet
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
So
Iwata
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[12579]
Open Access
Abstract: The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.
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Sep 2017
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I04-Macromolecular Crystallography
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Yilmaz
Alguel
,
Sotiris
Amillis
,
James
Leung
,
George
Lambrinidis
,
Stefano
Capaldi
,
Nicola J.
Scull
,
Gregory
Craven
,
So
Iwata
,
Alan
Armstrong
,
Emmanuel
Mikros
,
George
Diallinas
,
Alexander D.
Cameron
,
Bernadette
Byrne
Open Access
Abstract: The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.
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Apr 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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T.
Arakawa
,
T.
Kobayashi-Yurugi
,
Y.
Alguel
,
H.
Iwanari
,
H.
Hatae
,
M.
Iwata
,
Y.
Abe
,
T.
Hino
,
C.
Ikeda-Suno
,
H.
Kuma
,
D.
Kang
,
T.
Murata
,
T.
Hamakubo
,
A. D.
Cameron
,
T.
Kobayashi
,
N.
Hamasaki
,
S.
Iwata
Abstract: Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease.
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Nov 2015
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I02-Macromolecular Crystallography
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Norimichi
Nomura
,
Gregory
Verdon
,
Haejoo
Kang
,
Tatsuro
Shimamura
,
Yayoi
Nomura
,
Yo
Sonoda
,
Saba
Abdul Hussein
,
Aziz
Qureshi
,
Mathieu
Coincon
,
Yumi
Sato
,
Hitomi
Abe
,
Yoshiko
Nakada-Nakura
,
Tomoya
Hino
,
Takatoshi
Arakawa
,
Osamu
Kusano-Arai
,
Hiroko
Iwanari
,
Takeshi
Murata
,
Takuya
Kobayashi
,
Takao
Hamakubo
,
Michihiro
Kasahara
,
So
Iwata
,
David
Drew
Abstract: The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters.
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Oct 2015
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The structure determination of an integral membrane protein using synchrotron
X-ray diffraction data collected at room temperature directly in vapour diffusion
crystallization plates (in situ) is demonstrated. Exposing the crystals
in situ eliminates manual sample handling and, since it is performed at
room temperature, removes the complication of cryoprotection and potential
structural anomalies induced by sample cryocooling. Essential to the method is
the ability to limit radiation damage by recording a small amount of data per
sample from many samples and subsequently assembling the resulting data sets
using specialized software. The validity of this procedure is established by the
structure determination of Haemophilus influenza TehA at 2.3 A˚ resolution.
The method presented offers an effective protocol for the fast and efficient
determination of membrane-protein structures at room temperature using third generation
synchrotron beamlines.
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Jun 2015
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I24-Microfocus Macromolecular Crystallography
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Hiroaki
Tanabe
,
Yoshifumi
Fujii
,
Miki
Okada-Iwabu
,
Masato
Iwabu
,
Yoshihiro
Nakamura
,
Toshiaki
Hosaka
,
Kanna
Motoyama
,
Mariko
Ikeda
,
Motoaki
Wakiyama
,
Takaho
Terada
,
Noboru
Ohsawa
,
Masakatsu
Hato
,
Satoshi
Ogasawara
,
Tomoya
Hino
,
Takeshi
Murata
,
So
Iwata
,
Kunio
Hirata
,
Yoshiaki
Kawano
,
Masaki
Yamamoto
,
Tomomi
Kimura-Someya
Abstract: Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5′ AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure–function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.
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Apr 2015
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I24-Microfocus Macromolecular Crystallography
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Hiroaki
Tanabe
,
Kanna
Motoyama
,
Mariko
Ikeda
,
Motoaki
Wakiyama
,
Takaho
Terada
,
Noboru
Ohsawa
,
Toshiaki
Hosaka
,
Masakatsu
Hato
,
Yoshifumi
Fujii
,
Yoshihiro
Nakamura
,
Satoshi
Ogasawara
,
Tomoya
Hino
,
Takeshi
Murata
,
So
Iwata
,
Miki
Okada-Iwabu
,
Masato
Iwabu
,
Kunio
Hirata
,
Yoshiaki
Kawano
,
Masaki
Yamamoto
,
Tomomi
Kimura-Someya
,
Mikako
Shirouzu
,
Toshimasa
Yamauchi
,
Takashi
Kadowaki
,
Shigeyuki
Yokoyama
Open Access
Abstract: The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89–375) and AdipoR2Δ99 (residues 100–386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.
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Jan 2015
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.
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Nov 2014
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