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John H.
Beale
,
Rachel
Bolton
,
Stephen A.
Marshall
,
Emma V.
Beale
,
Stephen B.
Carr
,
Ali
Ebrahim
,
Tadeo
Moreno-chicano
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Ivo
Tews
,
Robin L.
Owen
Open Access
Abstract: Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.
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Dec 2019
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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
Open Access
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.
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Nov 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493, 19458]
Open Access
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.
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Sep 2019
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I24-Microfocus Macromolecular Crystallography
|
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
Diamond Proposal Number(s):
[14493]
Open Access
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.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
|
Open Access
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.
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Jul 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Open Access
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.
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Mar 2019
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Metrology
Optics
|
Open Access
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.
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Dec 2018
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I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[14493]
Open Access
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.
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Nov 2018
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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
Open Access
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.
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Oct 2018
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I24-Microfocus Macromolecular Crystallography
|
Sam
Horrell
,
Demet
Kekilli
,
Kakali
Sen
,
Robin L.
Owen
,
Florian S. N.
Dworkowski
,
Svetlana V.
Antonyuk
,
Thomas W.
Keal
,
Chin W.
Yong
,
Robert R.
Eady
,
S. Samar
Hasnain
,
Richard W.
Strange
,
Mike
Hough
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.
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May 2018
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