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.

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.

scholarly journals Computer-controlled liquid-nitrogen drizzling device for removing frost from cryopreserved crystals

2020 ◽  
Vol 76 (12) ◽  
pp. 616-622
Author(s):  
Yuki Nakamura ◽  
Seiki Baba ◽  
Nobuhiro Mizuno ◽  
Takaki Irie ◽  
Go Ueno ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Temperature Data ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Low Visibility ◽  
Computer Controlled

Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.

scholarly journals Room-temperature macromolecular serial crystallography using synchrotron radiation

IUCrJ ◽  
2014 ◽  
Vol 1 (4) ◽  
pp. 204-212 ◽  
Author(s):  
Francesco Stellato ◽  
Dominik Oberthür ◽  
Mengning Liang ◽  
Richard Bean ◽  
Cornelius Gati ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Single Crystal ◽  
Room Temperature ◽  
Structural Model ◽  
Three Dimensional ◽  
Single Crystal Diffraction ◽  
X Ray ◽  
Serial Crystallography ◽  
Diffraction Patterns

A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

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.

scholarly journals Anaerobic fixed-target serial crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (5) ◽  
pp. 901-912
Author(s):  
Patrick Rabe ◽  
John H. Beale ◽  
Agata Butryn ◽  
Pierre Aller ◽  
Anna Dirr ◽  
...  
Keyword(s):  
Data Collection ◽  
Room Temperature ◽  
Temperature Data ◽  
Protein Crystal ◽  
Light Sources ◽  
Strictly Anaerobic ◽  
X Ray ◽  
Synchrotron Light ◽  
Synchrotron Light Sources ◽  
Anaerobic Environment

Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.

scholarly journals Current status and future opportunities for serial crystallography at MAX IV Laboratory

2020 ◽  
Vol 27 (5) ◽  
pp. 1095-1102
Author(s):  
Anastasya Shilova ◽  
Hugo Lebrette ◽  
Oskar Aurelius ◽  
Jie Nan ◽  
Martin Welin ◽  
...  
Keyword(s):  
Free Electron ◽  
Room Temperature ◽  
High Viscosity ◽  
Current Status ◽  
Free Electron Lasers ◽  
X Ray ◽  
X Ray Crystallography ◽  
Injection Methods ◽  
Serial Crystallography ◽  
Presentation Methods

Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at  MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports – silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.

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.

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