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Instruments / Technical Area	Publication Details	Published
	High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography Tadeo Moreno Chicano , Ali Ebrahim , Danny Axford , Martin V. Appleby , John H. Beale , Amanda K. Chaplin , Helen M. E. Duyvesteyn , Reza A. Ghiladi , Shigeki Owada , Darren A. Sherrell , Richard Strange , Hiroshi Sugimoto , Kensuke Tono , Jonathan A. R. Worrall , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519011655 Open Access Show Abstract ▼ Abstract: High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.	Nov 2019
I24-Microfocus Macromolecular Crystallography	Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins Ali Ebrahim , Tadeo Moreno-chicano , Martin V. Appleby , Amanda K. Chaplin , John Beale , Darren A. Sherrell , Helen M. E. Duyvesteyn , Shigeki Owada , Kensuke Tono , Hiroshi Sugimoto , Richard W. Strange , Jonathan Worrall , Danny Axford , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519003956 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.	Jul 2019
	Crystallography on a chip – without the chip: sheet-on-sheet sandwich R. Bruce Doak , Gabriela Nass Kovacs , Alexander Gorel , Lutz Foucar , Thomas R. M. Barends , Marie Luise Grünbein , Mario Hilpert , Marco Kloos , Christopher M. Roome , Robert L. Shoeman , Miriam Stricker , Kensuke Tono , Daehyun You , Kiyoshi Ueda , Darren A. Sherrell , Robin Owen , Ilme Schlichting Acta Crystallographica Section D Structural Biology 74, 1000 - 1007 DOI: 10.1107/S2059798318011634 Open Access Show Abstract ▼ Abstract: Crystallography chips are fixed-target supports consisting of a film (for example Kapton) or wafer (for example silicon) that is processed using semiconductor-microfabrication techniques to yield an array of wells or through-holes in which single microcrystals can be lodged for raster-scan probing. Although relatively expensive to fabricate, chips offer an efficient means of high-throughput sample presentation for serial diffraction data collection at synchrotron or X-ray free-electron laser (XFEL) sources. Truly efficient loading of a chip (one microcrystal per well and no wastage during loading) is nonetheless challenging. The wells or holes must match the microcrystal size of interest, requiring that a large stock of chips be maintained. Raster scanning requires special mechanical drives to step the chip rapidly and with micrometre precision from well to well. Here, a `chip-less' adaptation is described that essentially eliminates the challenges of loading and precision scanning, albeit with increased, yet still relatively frugal, sample usage. The device consists simply of two sheets of Mylar with the crystal solution sandwiched between them. This sheet-on-sheet (SOS) sandwich structure has been employed for serial femtosecond crystallography data collection with micrometre-sized crystals at an XFEL. The approach is also well suited to time-resolved pump–probe experiments, in particular for long time delays. The SOS sandwich enables measurements under XFEL beam conditions that would damage conventional chips, as documented here. The SOS sheets hermetically seal the sample, avoiding desiccation of the sample provided that the X-ray beam does not puncture the sheets. This is the case with a synchrotron beam but not with an XFEL beam. In the latter case, desiccation, setting radially outwards from each punched hole, sets lower limits on the speed and line spacing of the raster scan. It is shown that these constraints are easily accommodated.	Oct 2018
	Time zero determination for FEL pump-probe studies based on ultrafast melting of bismuth S. W. Epp , M. Hada , Y. Zhong , Y. Kumagai , K. Motomura , S. Mizote , T. Ono , S. Owada , Danny Axford , S. Bakhtiarzadeh , H. Fukuzawa , Y. Hayashi , T. Katayama , A. Marx , H. M. Müller-werkmeister , R. L. Owen , Da. A. Sherrell , K. Tono , K. Ueda , F. Westermeier , R. J. D. Miller Structural Dynamics 4 DOI: 10.1063/1.4999701 Open Access Show Abstract ▼ Abstract: A common challenge for pump-probe studies of structural dynamics at X-ray free-electron lasers (XFELs) is the determination of time zero (T0)—the time an optical pulse (e.g., an optical laser) arrives coincidently with the probe pulse (e.g., a XFEL pulse) at the sample position. In some cases, T0 might be extracted from the structural dynamics of the sample's observed response itself, but generally, an independent robust method is required or would be superior to the inferred determination of T0. In this paper, we present how the structural dynamics in ultrafast melting of bismuth can be exploited for a quickly performed, reliable and accurate determination of T0 with a precision below 20 fs and an overall experimental accuracy of 50 fs to 150 fs (estimated). Our approach is potentially useful and applicable for fixed-target XFEL experiments, such as serial femtosecond crystallography, utilizing an optical pump pulse in the ultraviolet to near infrared spectral range and a pixelated 2D photon detector for recording crystallographic diffraction patterns in transmission geometry. In comparison to many other suitable approaches, our method is fairly independent of the pumping wavelength (UV–IR) as well as of the X-ray energy and offers a favorable signal contrast. The technique is exploitable not only for the determination of temporal characteristics of the experiment at the interaction point but also for investigating important conditions affecting experimental control such as spatial overlap and beam spot sizes.	Sep 2017
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

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