I24-Microfocus Macromolecular Crystallography
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Tadeo
Moreno-Chicano
,
Leiah M.
Carey
,
Danny
Axford
,
John H.
Beale
,
R. Bruce
Doak
,
Helen M. E.
Duyvesteyn
,
Ali
Ebrahim
,
Robert W.
Henning
,
Diana C. F.
Monteiro
,
Dean A.
Myles
,
Shigeki
Owada
,
Darren A.
Sherrell
,
Megan L.
Straw
,
Vukica
Šrajer
,
Hiroshi
Sugimoto
,
Kensuke
Tono
,
Takehiko
Tosha
,
Ivo
Tews
,
Martin
Trebbin
,
Richard W.
Strange
,
Kevin L.
Weiss
,
Jonathan A. R.
Worrall
,
Flora
Meilleur
,
Robin L.
Owen
,
Reza A.
Ghiladi
,
Michael A.
Hough
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.
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Sep 2022
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I24-Microfocus Macromolecular Crystallography
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Gyorgy
Babnigg
,
Darren A.
Sherrell
,
Youngchang
Kim
,
Jessica L.
Johnson
,
Boguslaw
Nocek
,
Kemin
Tan
,
Danny
Axford
,
Hui
Li
,
Lance
Bigelow
,
Lukas
Welk
,
Michael
Endres
,
Robin L.
Owen
,
Andrzej
Joachimiak
Abstract: Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre-sized droplets allow the very efficient use of protein samples and can produce large quantities of high-quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X-ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample-transport and data-collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50–500 pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data-collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X-ray beam by depositing an emulsion of droplets onto a silicon fixed-target serial device are provided. After three months of storage at 4°C, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample-delivery and data-collection strategy allows crystal droplets to be shipped and set aside until beamtime is available.
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Aug 2022
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P.
Mehrabi
,
R.
Bücker
,
G.
Bourenkov
,
H. M.
Ginn
,
D.
Von Stetten
,
H. M.
Müller-Werkmeister
,
A.
Kuo
,
T.
Morizumi
,
B.t.
Eger
,
W.-L.
Ou
,
S.
Oghbaey
,
A.
Sarracini
,
J. E.
Besaw
,
O.
Pare´-Labrosse
,
S.
Meier
,
H.
Schikora
,
F.
Tellkamp
,
A.
Marx
,
D. A.
Sherrell
,
D.
Axford
,
R. I.
Owen
,
O. P.
Ernst
,
E. F.
Pai
,
E. C.
Schulz
,
R. J. D.
Miller
Open Access
Abstract: For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.
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Mar 2021
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I24-Microfocus Macromolecular Crystallography
Data acquisition
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Open Access
Abstract: Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.
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Feb 2021
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Pedram
Mehrabi
,
Henrike
Mueller-Werkmeister
,
Jan-Philipp
Leimkohl
,
Hendrik
Schikora
,
Jelena
Ninkovic
,
Silvia
Krivokuca
,
Ladislav
Andriček
,
Sascha W.
Epp
,
Darren A.
Sherrell
,
Robin L.
Owen
,
Arwen R.
Pearson
,
Friedjof
Tellkamp
,
Eike
Schulz
,
R. J. Dwayne
Miller
Open Access
Abstract: Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.
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Mar 2020
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NONE-No attached Diamond beamline
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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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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
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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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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
Open Access
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.
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Sep 2017
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