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

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.

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Approach of Serial Crystallography

Crystals ◽

10.3390/cryst10100854 ◽

2020 ◽

Vol 10 (10) ◽

pp. 854

Author(s):

Ki Hyun Nam

Keyword(s):

Radiation Damage ◽

Room Temperature ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

The Past ◽

Wide Range ◽

Experimental Limitation ◽

Serial Crystallography ◽

Collection Strategies

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial.

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Lard Injection Matrix for Serial Crystallography

International Journal of Molecular Sciences ◽

10.3390/ijms21175977 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5977

Author(s):

Ki Hyun Nam

Keyword(s):

Flow Rate ◽

Room Temperature ◽

Glucose Isomerase ◽

Viscous Medium ◽

Cubic Phase ◽

Time Resolved ◽

Crystal Sample ◽

X Ray ◽

Serial Crystallography ◽

Delivery Medium

Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.

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Room-temperature macromolecular serial crystallography using synchrotron radiation

IUCrJ ◽

10.1107/s2052252514010070 ◽

2014 ◽

Vol 1 (4) ◽

pp. 204-212 ◽

Cited By ~ 151

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.

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Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Crystals ◽

10.3390/cryst10030215 ◽

2020 ◽

Vol 10 (3) ◽

pp. 215 ◽

Cited By ~ 7

Author(s):

Robert Cheng

Keyword(s):

Data Collection ◽

Collection Efficiency ◽

Structural Data ◽

Fast Time ◽

Full Potential ◽

Delivery Methods ◽

Time Resolved ◽

X Ray ◽

Drop On Demand ◽

Serial Crystallography

The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

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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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Segmented flow generator for serial crystallography at the European X-ray free electron laser

Nature Communications ◽

10.1038/s41467-020-18156-7 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Austin Echelmeier ◽

Jorvani Cruz Villarreal ◽

Marc Messerschmidt ◽

Daihyun Kim ◽

Jesse D. Coe ◽

...

Keyword(s):

Free Electron ◽

Structural Features ◽

Protein Crystal ◽

Liquid Sample ◽

Time Resolved ◽

X Ray ◽

Flow Generator ◽

Serial Crystallography ◽

Serial Femtosecond Crystallography

Abstract Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

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Millisecond time-resolved serial oscillation crystallography of a blue-light photoreceptor at a synchrotron

IUCrJ ◽

10.1107/s2052252520007411 ◽

2020 ◽

Vol 7 (4) ◽

pp. 728-736

Author(s):

Sylvain Aumonier ◽

Gianluca Santoni ◽

Guillaume Gotthard ◽

David von Stetten ◽

Gordon A. Leonard ◽

...

Keyword(s):

Blue Light ◽

Room Temperature ◽

Intermediate Species ◽

Time Resolved ◽

X Ray ◽

Blue Light Photoreceptor ◽

Intermediate States ◽

Serial Crystallography ◽

Protein Reaction ◽

Monochromatic Synchrotron

The recent development of serial crystallography has popularized time-resolved crystallography as a technique to determine the structure of protein-reaction intermediate states. However, most approaches rely on the availability of thousands to millions of microcrystals. A method is reported here, using monochromatic synchrotron radiation, for the room-temperature collection, processing and merging of X-ray oscillation diffraction data from <100 samples in order to observe the build up of a photoreaction intermediate species. Using this method, we monitored with a time resolution of 63 ms how the population of a blue-light photoreceptor domain in a crystal progressively photoconverts from the dark to the light state. The series of resulting snapshots allows us to visualize in detail the gradual rearrangement of both the protein and chromophore during this process.

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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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Serial X-ray Crystallography

Crystals ◽

10.3390/cryst12010099 ◽

2022 ◽

Vol 12 (1) ◽

pp. 99

Author(s):

Ki Hyun Nam

Keyword(s):

Room Temperature ◽

X Rays ◽

Time Resolved ◽

Pump Probe ◽

X Ray ◽

X Ray Crystallography ◽

Target Molecules ◽

Serial Crystallography ◽

Pump Probe Experiment ◽

Serial Femtosecond Crystallography

Serial crystallography (SX) is an emerging technique to determine macromolecules at room temperature. SX with a pump–probe experiment provides the time-resolved dynamics of target molecules. SX has developed rapidly over the past decade as a technique that not only provides room-temperature structures with biomolecules, but also has the ability to time-resolve their molecular dynamics. The serial femtosecond crystallography (SFX) technique using an X-ray free electron laser (XFEL) has now been extended to serial synchrotron crystallography (SSX) using synchrotron X-rays. The development of a variety of sample delivery techniques and data processing programs is currently accelerating SX research, thereby increasing the research scope. In this editorial, I briefly review some of the experimental techniques that have contributed to advances in the field of SX research and recent major research achievements. This Special Issue will contribute to the field of SX research.

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3D-printed holders for in meso in situ fixed-target serial X-ray crystallography

Journal of Applied Crystallography ◽

10.1107/s1600576720002897 ◽

2020 ◽

Vol 53 (3) ◽

pp. 854-859

Author(s):

Chia-Ying Huang ◽

Nathalie Meier ◽

Martin Caffrey ◽

Meitian Wang ◽

Vincent Olieric

Keyword(s):

Data Collection ◽

Heavy Atom ◽

Fixed Target ◽

X Ray ◽

X Ray Crystallography ◽

Sample Stability ◽

3D Printed ◽

Sponge Phase ◽

Serial Crystallography

The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient post-crystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

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