scholarly journals In vacuoX-ray data collection from graphene-wrapped protein crystals

2015 ◽  
Vol 71 (10) ◽  
pp. 2079-2088 ◽  
Author(s):  
Anna J. Warren ◽  
Adam D. Crawshaw ◽  
Jose Trincao ◽  
Pierre Aller ◽  
Simon Alcock ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Protein Crystals ◽  
Quality Data ◽  
Liquid Jets ◽  
High Quality Data ◽  
X Ray ◽  
Serial Data ◽  
X Ray Background ◽  
Low X

The measurement of diffraction data from macromolecular crystal samples heldin vacuoholds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

scholarly journals Automated harvesting and processing of protein crystals through laser photoablation

2016 ◽  
Vol 72 (4) ◽  
pp. 454-466 ◽  
Author(s):  
Ulrich Zander ◽  
Guillaume Hoffmann ◽  
Irina Cornaciu ◽  
Jean-Pierre Marquette ◽  
Gergely Papp ◽  
...  
Keyword(s):  
Data Collection ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
New Methods ◽  
Repeated Sample ◽  
Sample Recovery ◽  
Through Diffusion ◽  
X Ray Background ◽  
Low X

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

scholarly journals The rise of neutron cryo-crystallography

2018 ◽  
Vol 74 (8) ◽  
pp. 792-799 ◽  
Author(s):  
Hanna Kwon ◽  
Patricia S. Langan ◽  
Leighton Coates ◽  
Emma L. Raven ◽  
Peter C. E. Moody
Keyword(s):  
Data Collection ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Protein Crystallography ◽  
Protein Crystals ◽  
X Ray ◽  
Problems And Solutions ◽  
Neutron Protein Crystallography ◽  
Direct Extrapolation ◽  
Neutron Crystallography

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.

scholarly journals 3D-MiXD: 3D-printed X-ray-compatible microfluidic devices for rapid, low-consumption serial synchrotron crystallography data collection in flow

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 207-219 ◽  
Author(s):  
Diana C. F. Monteiro ◽  
David von Stetten ◽  
Claudia Stohrer ◽  
Marta Sans ◽  
Arwen R. Pearson ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Time Resolved ◽  
X Ray ◽  
Biologically Relevant ◽  
3D Printed ◽  
Serial Crystallography ◽  
Low X ◽  
Very High

Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.

A fibre-based crystal mounting technique for protein cryocrystallography

2012 ◽  
Vol 45 (2) ◽  
pp. 362-366 ◽  
Author(s):  
Michihiro Sugahara
Keyword(s):  
Data Collection ◽  
Success Rate ◽  
Single Crystals ◽  
Conventional Technique ◽  
Synthetic Zeolite ◽  
Quality Data ◽  
High Quality ◽  
High Quality Data ◽  
X Ray ◽  
Loop Technique

The CryoFibre, a crystal mounting tool, has been developed for protein cryocrystallography. The technique attaches single crystals to the tips of polyester fibres, allowing removal of excess liquid around each crystal. Single-wavelength anomalous dispersion phasing using a Cu Kα X-ray source (Cu SAD) was applied to crystals from five proteins without any derivatization, demonstrating a clear improvement in the success rate of Cu SAD compared with the conventional loop technique. In addition, a xylanase crystal on the surface of a synthetic zeolite as a hetero-epitaxic nucleant was directly mounted on the CryoFibre without separation treatment of the crystal from the zeolite. The crystal had a lower mosaicity than that observed using the conventional technique, indicating that the fibre technique is suitable for high-quality data collection from zeolite-mediated crystals.

scholarly journals An automated platform for in situ serial crystallography at room temperature

IUCrJ ◽  
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.

scholarly journals High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions

2018 ◽  
Vol 13 (2) ◽  
pp. 260-292 ◽  
Author(s):  
Jana Broecker ◽  
Takefumi Morizumi ◽  
Wei-Lin Ou ◽  
Viviane Klingel ◽  
Anling Kuo ◽  
...  
Keyword(s):  
Data Collection ◽  
High Throughput ◽  
Room Temperature ◽  
Protein Crystals ◽  
X Ray ◽  
Cryogenic Conditions

scholarly journals Low-dose fixed-target serial synchrotron crystallography

2017 ◽  
Vol 73 (4) ◽  
pp. 373-378 ◽  
Author(s):  
Robin L. Owen ◽  
Danny Axford ◽  
Darren A. Sherrell ◽  
Anling Kuo ◽  
Oliver P. Ernst ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Dose ◽  
Quality Data ◽  
Translation System ◽  
Fixed Target ◽  
High Quality Data ◽  
X Ray ◽  
Serial Sampling ◽  
Serial Data ◽  
Serial Crystallography

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

A simple and rapid method for mounting protein crystals at room temperature

2003 ◽  
Vol 36 (1) ◽  
pp. 165-166 ◽  
Author(s):  
Aengus Mac Sweeney ◽  
Allan D'Arcy
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Protein Crystals ◽  
Freezing Process ◽  
Routine Method ◽  
X Ray ◽  
Significant Damage ◽  
Simplified Method ◽  
Intrinsic Quality

Cryocooling of protein crystals for X-ray data collection has now become a routine method in the majority of biostructural laboratories. The improvement of facilities at synchrotron sources and their increased use has made it essential to have properly frozen crystals for optimal data collection. Although in general crystals can be cooled without significant damage, there are often cases in which crystals with slight disorder or twinning problems suffer considerably during the freezing process. In other cases, poor or mosaic diffraction may be blamed on the cryoprotectant or cooling protocol. Many crystals may be wasted in searching for the best freezing conditions when the intrinsic quality of the crystals is poor. In principle, the collection of room-temperature diffraction data would provide a reference that would allow the detection of crystal damage caused by addition of cryoprotectant or by cryocooling. In practice, however, many investigators are reluctant to do this, one reason being that capillary mounting of crystals is a tedious method, especially for those who are new to crystallography. Here a simplified method for mounting crystals at room temperature is reported, which requires little expertise and no expensive equipment.

scholarly journals Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams

2015 ◽  
Vol 71 (2) ◽  
pp. 387-397 ◽  
Author(s):  
Sabine Botha ◽  
Karol Nass ◽  
Thomas R. M. Barends ◽  
Wolfgang Kabsch ◽  
Beatrice Latz ◽  
...  
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
High Throughput ◽  
Room Temperature ◽  
High Viscosity ◽  
Temperature Data ◽  
High Dose ◽  
X Ray ◽  
Serial Data ◽  
Serial Crystallography

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These includein situcollection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allowde novostructure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.

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