I24-Microfocus Macromolecular Crystallography
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James
Birch
,
Tristan O. C.
Kwan
,
Peter J.
Judge
,
Danny
Axford
,
Pierre
Aller
,
Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
,
Javier
Vinals
,
Robin L.
Owen
,
Eriko
Nango
,
Rie
Tanaka
,
Kensuke
Tono
,
Yasumasa
Joti
,
Tomoyuki
Tanaka
,
Shigeki
Owada
,
Michihiro
Sugahara
,
So
Iwata
,
Allen M.
Orville
,
Anthony
Watts
,
Isabel
Moraes
Diamond Proposal Number(s):
[19152]
Open Access
Abstract: Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
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Oct 2023
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I03-Macromolecular Crystallography
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Christopher D. M.
Hutchison
,
James
Baxter
,
Ann
Fitzpatrick
,
Gabriel
Dorlhiac
,
Alisia
Fadini
,
Samuel
Perrett
,
Karim
Maghlaoui
,
Salomé
Bodet Lefèvre
,
Violeta
Cordon-Preciado
,
Josie L.
Ferreira
,
Volha U.
Chukhutsina
,
Douglas
Garratt
,
Jonathan
Barnard
,
Gediminas
Galinis
,
Flo
Glencross
,
Rhodri M.
Morgan
,
Sian
Stockton
,
Ben
Taylor
,
Letong
Yuan
,
Matthew G.
Romei
,
Chi-Yun
Lin
,
Jon P.
Marangos
,
Marius
Schmidt
,
Viktoria
Chatrchyan
,
Tiago
Buckup
,
Dmitry
Morozov
,
Jaehyun
Park
,
Sehan
Park
,
Intae
Eom
,
Minseok
Kim
,
Dogeun
Jang
,
Hyeongi
Choi
,
Hyojung
Hyun
,
Gisu
Park
,
Eriko
Nango
,
Rie
Tanaka
,
Shigeki
Owada
,
Kensuke
Tono
,
Daniel P.
Deponte
,
Sergio
Carbajo
,
Matt
Seaberg
,
Andrew
Aquila
,
Sebastien
Boutet
,
Anton
Barty
,
So
Iwata
,
Steven G.
Boxer
,
Gerrit
Groenhof
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[22819, 17221]
Open Access
Abstract: The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump–probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.
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Aug 2023
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I23-Long wavelength MX
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Alisia
Fadini
,
Christopher D. M.
Hutchison
,
Dmitry
Morozov
,
Jeffrey
Chang
,
Karim
Maghlaoui
,
Samuel
Perrett
,
Fangjia
Luo
,
Jeslyn C. X.
Kho
,
Matthew G.
Romei
,
R. Marc L.
Morgan
,
Christian
Orr
,
Violeta
Cordon-Preciado
,
Takaaki
Fujiwara
,
Nipawan
Nuemket
,
Takehiko
Tosha
,
Rie
Tanaka
,
Shigeki
Owada
,
Kensuke
Tono
,
So
Iwata
,
Steven G.
Boxer
,
Gerrit
Groenhof
,
Eriko
Nango
,
Jasper J.
Van Thor
Diamond Proposal Number(s):
[23620]
Open Access
Abstract: Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.
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Jul 2023
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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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