Time-resolved serial crystallography of bacteriorhodopsin using synchrotrons and X-ray lasers

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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Viscous hydrophilic injection matrices for serial crystallography

IUCrJ ◽

10.1107/s2052252517005140 ◽

2017 ◽

Vol 4 (4) ◽

pp. 400-410 ◽

Cited By ~ 38

Author(s):

Gabriela Kovácsová ◽

Marie Luise Grünbein ◽

Marco Kloos ◽

Thomas R. M. Barends ◽

Ramona Schlesinger ◽

...

Keyword(s):

Protein Crystallization ◽

High Viscosity ◽

Stream Velocity ◽

Cubic Phase ◽

Time Resolved ◽

Flow Rates ◽

Block Polymer ◽

X Ray ◽

Crystallization Conditions ◽

Serial Crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used forin mesomembrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydrogels as viscous injection matrices is described, namely sodium carboxymethyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.

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High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source

IUCrJ ◽

10.1107/s205225251900263x ◽

2019 ◽

Vol 6 (3) ◽

pp. 412-425 ◽

Cited By ~ 17

Author(s):

Jose M. Martin-Garcia ◽

Lan Zhu ◽

Derek Mendez ◽

Ming-Yue Lee ◽

Eugene Chun ◽

...

Keyword(s):

Synchrotron Radiation ◽

Radiation Source ◽

Structural Changes ◽

High Viscosity ◽

Synchrotron Radiation Source ◽

Proteinase K ◽

Moderate Increase ◽

Time Resolved ◽

X Ray ◽

Serial Crystallography

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth (`pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2A adenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.

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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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Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes

Lab on a Chip ◽

10.1039/d0lc00239a ◽

2020 ◽

Vol 20 (16) ◽

pp. 2954-2964

Author(s):

Mark A. Levenstein ◽

Yi-Yeoun Kim ◽

Liam Hunter ◽

Clara Anduix-Canto ◽

Carlos González Niño ◽

...

Keyword(s):

Crystal Growth ◽

Diffraction Analysis ◽

X Ray Diffraction ◽

Time Resolved ◽

X Ray ◽

Scale Inhibition ◽

Serial Crystallography

Exploration of crystal growth in microchannels led to effective scale inhibition, enabling time-resolved injector-based serial crystallography-on-a-chip.

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Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15009966 ◽

2015 ◽

Vol 71 (7) ◽

pp. 856-860 ◽

Cited By ~ 9

Author(s):

Wenting Wu ◽

Przemyslaw Nogly ◽

Jan Rheinberger ◽

Leonhard M. Kick ◽

Cornelius Gati ◽

...

Keyword(s):

Free Electron ◽

Free Electron Laser ◽

Second Harmonic ◽

Night Vision ◽

Salt Crystallization ◽

Time Resolved ◽

Batch Crystallization ◽

X Ray ◽

Vapour Diffusion ◽

Serial Crystallography

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

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Approach of Serial Crystallography II

Crystals ◽

10.3390/cryst11060655 ◽

2021 ◽

Vol 11 (6) ◽

pp. 655

Author(s):

Ki-Hyun Nam

Keyword(s):

Free Electron ◽

Free Electron Laser ◽

Cryogenic Temperature ◽

Research Trends ◽

Special Issue ◽

Time Resolved ◽

Pump Probe ◽

X Ray ◽

Fluctuation Dynamics ◽

Serial Crystallography

Serial crystallography (SX) is an emerging X-ray crystallographic method for determining macromolecule structures. It can address concerns regarding the limitations of data collected by conventional crystallography techniques, which require cryogenic-temperature environments and allow crystals to accumulate radiation damage. Time-resolved SX studies using the pump-probe methodology provide useful information for understanding macromolecular mechanisms and structure fluctuation dynamics. This Special Issue deals with the serial crystallography approach using an X-ray free electron laser (XFEL) and synchrotron X-ray source, and reviews recent SX research involving synchrotron use. These reports provide insights into future serial crystallography research trends and approaches.

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

IUCrJ ◽

10.1107/s2052252519016865 ◽

2020 ◽

Vol 7 (2) ◽

pp. 207-219 ◽

Cited By ~ 9

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.

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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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Sample Delivery Media for Serial Crystallography

International Journal of Molecular Sciences ◽

10.3390/ijms20051094 ◽

2019 ◽

Vol 20 (5) ◽

pp. 1094 ◽

Cited By ~ 19

Author(s):

Ki Nam

Keyword(s):

Radiation Damage ◽

Molecular Mechanisms ◽

Structural Information ◽

Interaction Point ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography ◽

Serial Crystallography ◽

Serial Femtosecond Crystallography ◽

Delivery Medium

X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.

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