How to minimize X-ray dose used for in-house data collection on protein crystals

The use of boiled-off liquid nitrogen to maintain protein crystals at 100 K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.

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Faculty Opinions recommendation of In vacuo X-ray data collection from graphene-wrapped protein crystals.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725846656.793514045 ◽

2016 ◽

Author(s):

Anastassis Perrakis

Keyword(s):

Data Collection ◽

Protein Crystals ◽

X Ray

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A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913009700 ◽

2013 ◽

Vol 69 (7) ◽

pp. 1223-1230 ◽

Cited By ~ 72

Author(s):

Igor Nederlof ◽

Eric van Genderen ◽

Yao-Wang Li ◽

Jan Pieter Abrahams

Keyword(s):

Electron Diffraction ◽

Data Collection ◽

Diffraction Data ◽

Three Dimensional ◽

Protein Crystal ◽

Protein Crystals ◽

Electron Diffraction Data ◽

Single Shot ◽

X Ray ◽

Area Detector

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

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A hands-free capillary system for protein crystallography from crystallization to data collection

Journal of Applied Crystallography ◽

10.1107/s0021889808035772 ◽

2008 ◽

Vol 42 (1) ◽

pp. 129-133

Author(s):

Michihiro Sugahara ◽

Yuichi Kageyama ◽

Naoki Kunishima

Keyword(s):

Data Collection ◽

Diffraction Data ◽

Thermus Thermophilus ◽

Heavy Atom ◽

Experimental System ◽

Protein Crystals ◽

Glass Capillary ◽

Free System ◽

X Ray ◽

Capillary System

With the goal of producing a fully automated experimental system for protein X-ray crystallography, a hands-free system is presented in which all aspects of crystallographic experiments, from crystallization to diffraction data collection, are performed within a modified X-ray glass capillary. Because it eliminates the manual handling of crystals, this capillary system allows the user to evaluate the quality of protein crystals more accurately. The capabilities of this capillary system were examined using the TTHB049 protein fromThermus thermophilusHB8 and xylanase fromTrichoderma longibrachiatum. Each protein was successfully crystallized in the capillary and three complete diffraction data sets were collected at 100 K without direct manual intervention. The diffraction data from the capillary system were superior, in terms of both quality and reproducibility, when compared with data from conventional cryoloop systems. In addition, the capillary system allows thein situheavy-atom derivatization of protein crystals,i.e.the TTHB049 crystals were successfully derivatized with K2PtCl4within the capillary.

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A multiple-counter X-ray diffractometer with equatorial geometry

Journal of Applied Crystallography ◽

10.1107/s0021889877012783 ◽

1977 ◽

Vol 10 (1) ◽

pp. 45-51 ◽

Cited By ~ 11

Author(s):

D. W. Banner ◽

P. R. Evans ◽

D. J. Marsh ◽

D. C. Phillips

Keyword(s):

Data Collection ◽

Single Crystals ◽

Protein Crystals ◽

X Ray ◽

Design Construction

The design, construction and operation of a multiple-counter diffractometer are described. The instrument is used to measure up to five X-ray reflections from single crystals quasi-simultaneously: rates of data collection from protein crystals of 250 reflections per hour are achieved.

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Two-stage robotic crystal mounting of protein crystals for X-ray data collection

2008 IEEE International Conference on Automation Science and Engineering ◽

10.1109/coase.2008.4626573 ◽

2008 ◽

Author(s):

Atanas Georgiev ◽

Peter K. Allen

Keyword(s):

Data Collection ◽

Protein Crystals ◽

Two Stage ◽

X Ray

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Visualization of protein crystals by high-energy phase-contrast X-ray imaging

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319011379 ◽

2019 ◽

Vol 75 (11) ◽

pp. 947-958 ◽

Cited By ~ 4

Author(s):

Maxim Polikarpov ◽

Gleb Bourenkov ◽

Irina Snigireva ◽

Anatoly Snigirev ◽

Sophie Zimmermann ◽

...

Keyword(s):

Synchrotron Radiation ◽

Data Collection ◽

Diffraction Data ◽

Phase Contrast ◽

High Energy ◽

Protein Crystals ◽

Cubic Phase ◽

Macromolecular Crystallography ◽

X Ray ◽

X Ray Imaging

For the extraction of the best possible X-ray diffraction data from macromolecular crystals, accurate positioning of the crystals with respect to the X-ray beam is crucial. In addition, information about the shape and internal defects of crystals allows the optimization of data-collection strategies. Here, it is demonstrated that the X-ray beam available on the macromolecular crystallography beamline P14 at the high-brilliance synchrotron-radiation source PETRA III at DESY, Hamburg, Germany can be used for high-energy phase-contrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix. Three-dimensional tomograms have been obtained at X-ray doses that are substantially smaller and on time scales that are substantially shorter than those used for diffraction-scanning approaches that display protein crystals at micrometre resolution. Adding a compound refractive lens as an objective to the imaging setup, two-dimensional imaging at sub-micrometre resolution has been achieved. All experiments were performed on a standard macromolecular crystallography beamline and are compatible with standard diffraction data-collection workflows and apparatus. Phase-contrast X-ray imaging of macromolecular crystals could find wide application at existing and upcoming low-emittance synchrotron-radiation sources.

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X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444912002946 ◽

2012 ◽

Vol 68 (5) ◽

pp. 505-510 ◽

Cited By ~ 7

Author(s):

Robin L. Owen ◽

Briony A. Yorke ◽

Arwen R. Pearson

Keyword(s):

Data Collection ◽

Radiation Damage ◽

Diffraction Data ◽

Protein Crystals ◽

X Ray ◽

Optical Luminescence

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An automated platform for in situ serial crystallography at room temperature

IUCrJ ◽

10.1107/s2052252520011288 ◽

2020 ◽

Vol 7 (6) ◽

pp. 1009-1018

Author(s):

Zhong Ren ◽

Cong Wang ◽

Heewhan Shin ◽

Sepalika Bandara ◽

Indika Kumarapperuma ◽

...

Keyword(s):

Data Collection ◽

Room Temperature ◽

Protein Structures ◽

Routine Data ◽

Protein Crystals ◽

X Ray Diffraction ◽

X Ray ◽

Serial Crystallography ◽

Routine Data Collection

Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. This platform has been tested extensively using fragile protein crystals. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.

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