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

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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A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

Crystal Growth & Design ◽

10.1021/acs.cgd.6b00950 ◽

2016 ◽

Vol 16 (11) ◽

pp. 6318-6326 ◽

Cited By ~ 19

Author(s):

Jana Broecker ◽

Viviane Klingel ◽

Wei-Lin Ou ◽

Aidin R. Balo ◽

David J. Kissick ◽

...

Keyword(s):

Data Collection ◽

Membrane Protein ◽

High Throughput ◽

Protein Crystals ◽

X Ray

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The rise of neutron cryo-crystallography

Acta Crystallographica Section D Structural Biology ◽

10.1107/s205979831800640x ◽

2018 ◽

Vol 74 (8) ◽

pp. 792-799 ◽

Cited By ~ 5

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.

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Chapter 1. Practical Approaches for In Situ X-ray Crystallography: from High-throughput Screening to Serial Data Collection

Protein Crystallography - Chemical Biology ◽

10.1039/9781788010504-00001 ◽

2018 ◽

pp. 1-27 ◽

Cited By ~ 1

Author(s):

Isabelle Martiel ◽

Vincent Olieric ◽

Martin Caffrey ◽

Meitian Wang

Keyword(s):

Data Collection ◽

High Throughput ◽

High Throughput Screening ◽

X Ray ◽

X Ray Crystallography ◽

Serial Data

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A geometrical approach for semi-automated crystal centering andin situX-ray diffraction data collection

Journal of Applied Crystallography ◽

10.1107/s002188981301008x ◽

2013 ◽

Vol 46 (3) ◽

pp. 740-745 ◽

Cited By ~ 6

Author(s):

Mohammad Yaser Heidari Khajepour ◽

Hugo Lebrette ◽

Xavier Vernede ◽

Pierrick Rogues ◽

Jean-Luc Ferrer

Keyword(s):

Data Collection ◽

High Throughput ◽

Local Geometry ◽

Geometrical Approach ◽

Robot Arm ◽

X Ray Diffraction ◽

X Ray ◽

Analysis Process ◽

Handling Device

High-throughput protein crystallography projects pushed forward the development of automated crystallization platforms that are now commonly used. This created an urgent need for adapted and automated equipment for crystal analysis. However, first these crystals have to be harvested, cryo-protected and flash-cooled, operations that can fail or negatively impact on the crystal.In situX-ray diffraction analysis has become a valid alternative to these operations, and a growing number of users apply it for crystal screening and to solve structures. Nevertheless, even this shortcut may require a significant amount of beam time. In thisin situhigh-throughput approach, the centering of crystals relative to the beam represents the bottleneck in the analysis process. In this article, a new method to accelerate this process, by recording accurately the local geometry coordinates for each crystal in the crystallization plate, is presented. Subsequently, the crystallization plate can be presented to the X-ray beam by an automated plate-handling device, such as a six-axis robot arm, for an automated crystal centering in the beam,in situscreening or data collection. Here the preliminary results of such a semi-automated pipeline are reported for two distinct test proteins.

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In situX-ray crystallography

Journal of Applied Crystallography ◽

10.1107/s0021889800001254 ◽

2000 ◽

Vol 33 (2) ◽

pp. 397-400 ◽

Cited By ~ 18

Author(s):

Alexander McPherson

Keyword(s):

Data Collection ◽

Single Crystals ◽

Room Temperature ◽

Automated Data Collection ◽

Vapor Diffusion ◽

X Ray

A method is proposed, and preliminary experiments are described, for collection of X-ray data from macromolecular crystalsin situ. The usual processes of mounting for either room-temperature or cryogenic X-ray data collection are eliminated by growing crystals, using vapor diffusion, on small supports or films that can be either frozen or treated before transfer directly to the X-ray beam. The approach has the advantage that individual crystals are never manipulated and it is not necessary to isolate single crystals. Furthermore, crystals fixed to the surface on which they grow provides a positive advantage, small and otherwise problematic crystals become serviceable, and robotic or automated data collection becomes simplified.

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Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320008475 ◽

2020 ◽

Vol 76 (8) ◽

pp. 751-758

Author(s):

Jose A. Gavira ◽

Isaac Rodriguez-Ruiz ◽

Sergio Martinez-Rodriguez ◽

Shibom Basu ◽

Sébastien Teychené ◽

...

Keyword(s):

Data Collection ◽

Diffraction Data ◽

Room Temperature ◽

Low Cost ◽

Atomic Resolution ◽

Microfluidic Channels ◽

High Symmetry ◽

X Ray ◽

Counter Diffusion

Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of diffraction data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct data collection, and structure determination at atomic resolution close to 1.0 Å, is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution diffraction data collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete data sets at high resolution, the potential of in-line data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed.

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Using cryoloops for X-ray data collection from protein crystals at room temperature: A simple applicable method

Journal of Crystal Growth ◽

10.1016/j.jcrysgro.2005.04.047 ◽

2005 ◽

Vol 281 (2-4) ◽

pp. 592-595 ◽

Cited By ~ 1

Author(s):

Shu Jie Li ◽

Mamoru Suzuki ◽

Atsushi Nakagawa

Keyword(s):

Data Collection ◽

Room Temperature ◽

Protein Crystals ◽

X Ray

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

Journal of Applied Crystallography ◽

10.1107/s0021889802019623 ◽

2003 ◽

Vol 36 (1) ◽

pp. 165-166 ◽

Cited By ~ 6

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.

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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714026327 ◽

2015 ◽

Vol 71 (2) ◽

pp. 387-397 ◽

Cited By ~ 129

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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