Sample delivery for serial crystallography at free-electron lasers and synchrotrons

The high peak brilliance and femtosecond pulse duration of X-ray free-electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high-throughput serial approach to sample delivery. To this end, a number of such sample-delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low-dose data collection at room temperature. Here, the current sample-delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high-viscosity extrusion, including their application for time-resolved experiments. The challenges associated with sample delivery at megahertz repetition-rate XFELs are also outlined.

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Pump-Probe Time-Resolved Serial Femtosecond Crystallography at SACLA: Current Status and Data Collection Strategies

Applied Sciences ◽

10.3390/app9245505 ◽

2019 ◽

Vol 9 (24) ◽

pp. 5505 ◽

Cited By ~ 3

Author(s):

Eriko Nango ◽

Minoru Kubo ◽

Kensuke Tono ◽

So Iwata

Keyword(s):

Data Collection ◽

Free Electron ◽

Free Electron Laser ◽

Current Status ◽

Optical Pump ◽

Time Resolved ◽

Pump Probe ◽

X Ray ◽

Collection Strategies ◽

Serial Femtosecond Crystallography

Structural information on protein dynamics is a critical factor in fully understanding the protein functions. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) is a recently established technique for visualizing the structural changes or reactions in proteins that are at work with high spatial and temporal resolution. In the pump-probe method, protein microcrystals are continuously delivered from an injector and exposed to an X-ray free-electron laser (XFEL) pulse after a trigger to initiate a reaction, such as light, chemicals, temperature, and electric field, which affords the structural snapshots of intermediates that occur in the protein. We are in the process of developing the device and techniques for pump-probe TR-SFX while using XFEL produced at SPring-8 Angstrom Compact Free-Electron Laser (SACLA). In this paper, we described our current development details and data collection strategies for the optical pump X-ray probe TR-SFX experiment at SACLA and then reported the techniques of in crystallo TR spectroscopy, which is useful in clarifying the nature of reaction that takes place in crystals in advance.

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Pump-Probe Time-Resolved Serial Femtosecond Crystallography at X-Ray Free Electron Lasers

Crystals ◽

10.3390/cryst10070628 ◽

2020 ◽

Vol 10 (7) ◽

pp. 628

Author(s):

Suraj Pandey ◽

Ishwor Poudyal ◽

Tek Narsingh Malla

Keyword(s):

Free Electron ◽

Reaction Dynamics ◽

Reaction Coordinate ◽

Biological Macromolecules ◽

Free Electron Lasers ◽

Time Resolved ◽

Pump Probe ◽

X Ray ◽

Recent Developments ◽

Serial Femtosecond Crystallography

With time-resolved crystallography (TRX), it is possible to follow the reaction dynamics in biological macromolecules by investigating the structure of transient states along the reaction coordinate. X-ray free electron lasers (XFELs) have enabled TRX experiments on previously uncharted femtosecond timescales. Here, we review the recent developments, opportunities, and challenges of pump-probe TRX at XFELs.

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Versatile microporous polymer-based supports for serial macromolecular crystallography

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321007324 ◽

2021 ◽

Vol 77 (9) ◽

pp. 1153-1167

Author(s):

Isabelle Martiel ◽

John H. Beale ◽

Agnieszka Karpik ◽

Chia-Ying Huang ◽

Laura Vera ◽

...

Keyword(s):

Data Collection ◽

Free Electron ◽

Light Sources ◽

Free Electron Lasers ◽

Thin Specimen ◽

Screening Experiments ◽

Wide Range ◽

Serial Data ◽

Attractive Option ◽

Dose Limits

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

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Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0318 ◽

2014 ◽

Vol 369 (1647) ◽

pp. 20130318 ◽

Cited By ~ 43

Author(s):

Richard Neutze

Keyword(s):

Structural Dynamics ◽

Free Electron ◽

Free Electron Lasers ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

Protein Structural Dynamics ◽

Structural Biochemistry ◽

Scientific Questions ◽

Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10 12 X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.

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Time-Resolved Serial Femtosecond Crystallography at the European X-ray Free Electron Laser

10.21203/rs.2.14634/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marius Schmidt ◽

Suraj Pandey ◽

Adrian Mancuso ◽

Richard Bean

Keyword(s):

Free Electron ◽

Free Electron Laser ◽

Crystallographic Data ◽

Time Resolved ◽

X Ray ◽

Serial Femtosecond Crystallography

Abstract This protocol introduces step by step into the collection of time resolved crystallographic data and their analysis at the European Free Electron Laser.

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Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources

International Journal of Molecular Sciences ◽

10.3390/ijms20061401 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1401 ◽

Cited By ~ 13

Author(s):

Marius Schmidt

Keyword(s):

Structural Biology ◽

Free Electron ◽

Reaction Dynamics ◽

X Rays ◽

Free Electron Lasers ◽

Macromolecular Crystallography ◽

Time Resolved ◽

X Ray ◽

Methodological Aspects

The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.

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Liquid sample delivery techniques for serial femtosecond crystallography

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0337 ◽

2014 ◽

Vol 369 (1647) ◽

pp. 20130337 ◽

Cited By ~ 73

Author(s):

Uwe Weierstall

Keyword(s):

Flow Rate ◽

Free Electron ◽

Repetition Rate ◽

Free Electron Laser ◽

Room Temperature ◽

Liquid Flow Rate ◽

Free Electron Lasers ◽

Liquid Sample ◽

X Ray ◽

Serial Femtosecond Crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.

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