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54 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography 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 Journal Of Applied Crystallography 56 DOI: 10.1107/S1600576723006428 Diamond Proposal Number(s): [19152] Open Access Show Abstract ▼ 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.	Oct 2023
I03-Macromolecular Crystallography	Optical control of ultrafast structural dynamics in a fluorescent protein 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 Nature Chemistry 4 DOI: 10.1038/s41557-023-01275-1 Diamond Proposal Number(s): [22819, 17221] Open Access Show Abstract ▼ 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.	Aug 2023
I23-Long wavelength MX	Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism 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 Journal Of The American Chemical Society DOI: 10.1021/jacs.3c02313 Diamond Proposal Number(s): [23620] Open Access Show Abstract ▼ 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.	Jul 2023
Krios I-Titan Krios I at Diamond	Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides 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 Science Advances 7 DOI: 10.1126/sciadv.abj5363 Diamond Proposal Number(s): [18659] Open Access Show Abstract ▼ 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.	Sep 2021
	Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors Tetsuo Yamashita , Daniel Ken Inaoka , Tomoo Shiba , Takumi Oohashi , So Iwata , Takao Yagi , Hiroaki Kosaka , Hideto Miyoshi , Shigeharu Harada , Kiyoshi Kita , Katsuya Hirano Scientific Reports 8 DOI: 10.1038/s41598-018-20775-6 Open Access Show Abstract ▼ 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.	Feb 2018
B22-Multimode InfraRed imaging And Microspectroscopy I03-Macromolecular Crystallography I04-Macromolecular Crystallography	X-ray free electron laser determination of crystal structures of dark and light states of a reversibly photoswitching fluorescent protein at room temperature 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 International Journal Of Molecular Sciences 18 DOI: 10.3390/ijms18091918 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ 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.	Sep 2017
I04-Macromolecular Crystallography	Structure of eukaryotic purine/H+ symporter UapA suggests a role for homodimerization in transport activity 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 Nature Communications 7 DOI: 10.1038/ncomms11336 Open Access Show Abstract ▼ 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.	Apr 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Crystal structure of the anion exchanger domain of human erythrocyte band 3 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 Science 350 (6261), 680 - 684 DOI: 10.1126/science.aaa4335 PMID: 26542571 Show Abstract ▼ 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.	Nov 2015
I02-Macromolecular Crystallography	Structure and mechanism of the mammalian fructose transporter GLUT5 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 Nature 526, 397 - 401 DOI: 10.1038/nature14909 PMID: 26416735 Show Abstract ▼ 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.	Oct 2015
I24-Microfocus Macromolecular Crystallography	Structure determination of an integral membrane protein at room temperature from crystals in situ Danny Axford , James Foadi , Nien-Jen Hu , Hassan Choudhury , So Iwata , Konstantinos Beis , Gwyndaf Evans , Yilmaz Alguel Acta Crystallographica Section D Biological Crystallography 71 DOI: 10.1107/S139900471500423X PMID: 26057664 Open Access Show Abstract ▼ 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.	Jun 2015

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