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

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	Reducing sample consumption for serial crystallography using acoustic drop ejection Bradley Davy , Danny Axford , John H. Beale , Agata Butryn , Peter Docker , Ali Ebrahim , Gabriel Leen , Allen M. Orville , Robin L. Owen , Pierre Aller Journal Of Synchrotron Radiation 26 DOI: 10.1107/S1600577519009329 Diamond Proposal Number(s): [14493, 19458] Open Access Show Abstract ▼ Abstract: Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.	Sep 2019
I24-Microfocus Macromolecular Crystallography	Non-contact universal sample presentation for room temperature macromolecular crystallography using acoustic levitation R. H. Morris , E. R. Dye , D. Axford , M. I. Newton , J. H. Beale , P. T. Docker Scientific Reports 9 DOI: 10.1038/s41598-019-48612-4 Open Access Show Abstract ▼ Abstract: Macromolecular Crystallography is a powerful and valuable technique to assess protein structures. Samples are commonly cryogenically cooled to minimise radiation damage effects from the X-ray beam, but low temperatures hinder normal protein functions and this procedure can introduce structural artefacts. Previous experiments utilising acoustic levitation for beamline science have focused on Langevin horns which deliver significant power to the confined droplet and are complex to set up accurately. In this work, the low power, portable TinyLev acoustic levitation system is used in combination with an approach to dispense and contain droplets, free of physical sample support to aid protein crystallography experiments. This method facilitates efficient X-ray data acquisition in ambient conditions compatible with dynamic studies. Levitated samples remain free of interference from fixed sample mounts, receive negligible heating, do not suffer significant evaporation and since the system occupies a small volume, can be readily installed at other light sources.	Aug 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
I24-Microfocus Macromolecular Crystallography	Direct measurement of X-ray-induced heating of microcrystals Anna Warren , Danny Axford , Robin L. Owen Journal Of Synchrotron Radiation 26 DOI: 10.1107/S1600577519003849 Open Access Show Abstract ▼ Abstract: Temperature control is a key aspect of macromolecular crystallography, with the technique of cryocooling routinely being used to mitigate X-ray-induced damage. Beam-induced heating could cause the temperature of crystals to rise above the glass transition temperature, greatly increasing the rate of damage. X-ray-induced heating of ruby crystals of 20–40 µm in size has been quantified non-invasively by monitoring the emission wavelengths of X-ray-induced fluorescence during exposure to the X-ray beam. For the beam sizes and dose rates typically used in macromolecular crystallography, the temperature rises are of the order of 20 K. The temperature changes observed are compared with models in the literature and can be used as a validation tool for future models.	Jul 2019
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	How best to use photons Graeme Winter , Richard J. Gildea , Neil G. Paterson , John Beale , Markus Gerstel , Danny Axford , Melanie Vollmar , Katherine E. Mcauley , Robin L. Owen , Ralf Flaig , Alun W. Ashton , David Hall Acta Crystallographica Section D Structural Biology 75, 242 - 261 DOI: 10.1107/S2059798319003528 Open Access Show Abstract ▼ Abstract: Strategies for collecting X-ray diffraction data have evolved alongside beamline hardware and detector developments. The traditional approaches for diffraction data collection have emphasised collecting data from noisy integrating detectors (i.e. film, image plates and CCD detectors). With fast pixel array detectors on stable beamlines, the limiting factor becomes the sample lifetime, and the question becomes one of how to expend the photons that your sample can diffract, i.e. as a smaller number of stronger measurements or a larger number of weaker data. This parameter space is explored via experiment and synthetic data treatment and advice is derived on how best to use the equipment on a modern beamline. Suggestions are also made on how to acquire data in a conservative manner if very little is known about the sample lifetime.	Mar 2019
Metrology Optics	Dynamic adaptive X-ray optics. Part II. High-speed piezoelectric bimorph deformable Kirkpatrick–Baez mirrors for rapid variation of the 2D size and shape of X-ray beams Simon G. Alcock , Ioana-theodora Nistea , Riccardo Signorato , Robin L. Owen , Danniel Axford , John P. Sutter , Andrew Foster , Kawal Sawhney Journal Of Synchrotron Radiation 26 DOI: 10.1107/S1600577518015965 Open Access Show Abstract ▼ Abstract: The tangential curvature of actively bent X-ray mirrors at synchrotron radiation and X-ray free-electron laser (XFEL) facilities is typically only changed every few hours or even days. This operation can take tens of minutes for active optics with multiple bending actuators and often requires expert guidance using in situ monitoring devices. Hence, the dynamic performance of active X-ray optics for synchrotron beamlines has historically not been exploited. This is in stark contrast to many other scientific fields. However, many areas of synchrotron radiation and XFEL science, including macromolecular crystallography, could greatly benefit from the ability to change the size and shape of the X-ray beam rapidly and continuously. The advantages of this innovative approach are twofold: a large reduction in the dead time required to change the size of the X-ray beam for different-sized samples and the possibility of making multiple changes to the beam during the measurement of a single sample. In the preceding paper [Part I; Alcock, Nistea, Signorato & Sawhney (2019[Alcock, S. G., Nistea, I.-T., Signorato, R. & Sawhney, K. (2019). J. Synchrotron Rad. 26, this issue.]), J. Synchrotron Rad. 26, this issue], which accompanies this article, high-speed visible-light Fizeau interferometry was used to identify the factors which influence the dynamic bending behaviour of piezoelectric bimorph deformable X-ray mirrors. Building upon this ex situ metrology study, provided here is the first synchrotron radiation beamline implementation of high-speed adaptive X-ray optics using two bimorphs operating as a Kirkpatrick–Baez pair. With optimized substrates, novel opto-mechanical holders and a next-generation high-voltage power supply, the size of an X-ray beam was rapidly and repeatedly switched in <10 s. Of equal importance, it is also shown that compensation of piezoelectric creep ensures that the X-ray beam size remains stable for more than 1 h after making a major change. The era of high-speed adaptive X-ray optics for synchrotron radiation and XFEL beamlines has begun.	Dec 2018
I24-Microfocus Macromolecular Crystallography	Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography Ali Ebrahim , Martin V. Appleby , Danny Axford , John Beale , Tadeo Moreno-chicano , Darren A. Sherrell , Richard W. Strange , Michael A. Hough , Robin L. Owen Acta Crystallographica Section D Structural Biology 75 DOI: 10.1107/S2059798318010240 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.	Nov 2018
	The fine art of integral membrane protein crystallisation James Birch , Danny Axford , James Foadi , Arne Meyer , Annette Eckardt , Yvonne Thielmann , Isabel Moraes Methods DOI: 10.1016/j.ymeth.2018.05.014 Show Abstract ▼ Abstract: Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.	May 2018
I24-Microfocus Macromolecular Crystallography	An Acoustic On-Chip Goniometer for Room Temperature Macromolecular Crystallography C. G. Burton , Danny Axford , Andrew Michael John Edwards , Richard J. Gildea , Robert H. Morris , Michael I. Newton , Allen M. Orville , Mark Prince , Paul D. Topham , Peter D. Docker Lab On A Chip DOI: 10.1039/C7LC00812K Show Abstract ▼ Abstract: This paper describes the design, development and successful use of an on-chip goniometer for room-temperature macromolecular crystallography via acoustically induced rotations. We present for the first time a low cost, rate-tunable, acoustic actuator for randomised in-fluid sample orientation and its utilisation for protein structure determination on a synchrotron beamline. The device enables the efficient collection of diffraction data via a rotation method from a sample within a surface confined droplet. This method facillitates efficient macromolecular structural data acquisition in fluid environments for dynamical studies.	Nov 2017

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