scholarly journals Discerning best practices in XFEL-based biological crystallography – standards for nonstandard experiments

IUCrJ ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 532-543
Author(s):  
Alexander Gorel ◽  
Ilme Schlichting ◽  
Thomas R. M. Barends
Keyword(s):  
Best Practices ◽  
Structural Biology ◽  
Conformational Changes ◽  
Critical Evaluation ◽  
Quality Standards ◽  
Free Electron Lasers ◽  
High Resolution Data ◽  
X Ray ◽  
Serial Femtosecond Crystallography ◽  
Resolution Data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than `standard' crystallographic data. In contrast with this, the studies performed at XFELs often aim to investigate issues that require precise high-resolution data, for example to determine structures of intermediates at low occupancy, which often display very small conformational changes. This is a potentially dangerous combination and underscores the need for a critical evaluation of procedures including data-quality standards in XFEL-based structural biology. Here, such concerns are addressed.

scholarly journals Preventing Bio-Bloopers and XFEL Follies: Best Practices from your Friendly Instrument Staff

Crystals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 251 ◽  
Author(s):  
Christopher Kupitz ◽  
Raymond G. Sierra
Keyword(s):  
Best Practices ◽  
Structural Biology ◽  
High Speed ◽  
X Ray ◽  
Experimental Planning ◽  
The Past ◽  
Delivery Technique ◽  
Scientific Goal ◽  
Paradigm Shifting ◽  
Serial Femtosecond Crystallography

Serial Femtosecond Crystallography (SFX) at X-ray Free electron Lasers (XFELs) is a relatively new field promising to deliver unparalleled spatial and temporal resolution on biological systems and there dynamics. Over the past decade, though, there have been a handful of results that have truly delivered on these promises. Why? SFX has many paradigm shifting techniques not seen in typical structural biology arenas, such as creating a concentrated slurry of microcrystals rather than a handful of loopable prisms worthy of a catalog photo. Then taking that slurry and high speed jetting them towards the vacuum or helium interation region to destroy less than 1% of your sample and waste the other 99. The literature is full of techniques and methods promising to cure what ails your experiment, yet as an instrument scientist will tell you –and a first author might admit after a few drinks at the conference happy hour—is that there are a lot more failures than the success we published, results may vary. We will walk through a best practices on how to prepare your sample and chose a sample delivery technique that will amerliorate your studies rather than undermine your hardwork and hopefully lead to better experimental planning and execution, inching you closer to that scientific goal and that call from Stockholm. This will be written in a more editorialized fashion and is meant to give the reader an idea of what to try or how they should be thinking. Welcome to SFX, now what?

scholarly journals Membrane protein structural biology using X-ray free electron lasers

2015 ◽  
Vol 33 ◽  
pp. 115-125 ◽  
Author(s):  
Richard Neutze ◽  
Gisela Brändén ◽  
Gebhard FX Schertler
Keyword(s):  
Membrane Protein ◽  
Structural Biology ◽  
Free Electron ◽  
Free Electron Lasers ◽  
X Ray

scholarly journals Time-Resolved Macromolecular Crystallography at Pulsed X-ray Sources

2019 ◽  
Vol 20 (6) ◽  
pp. 1401 ◽  
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.

scholarly journals Liquid sample delivery techniques for serial femtosecond crystallography

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130337 ◽  
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.

scholarly journals In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130497 ◽  
Author(s):  
François-Xavier Gallat ◽  
Naohiro Matsugaki ◽  
Nathan P. Coussens ◽  
Koichiro J. Yagi ◽  
Marion Boudes ◽  
...  
Keyword(s):  
Structural Biology ◽  
Free Electron ◽  
Free Electron Laser ◽  
Protein Crystal ◽  
X Ray ◽  
Sample Characterization ◽  
The One ◽  
Serial Femtosecond Crystallography

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

scholarly journals Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA

2017 ◽  
Vol 24 (5) ◽  
pp. 1086-1091 ◽  
Author(s):  
Minoru Kubo ◽  
Eriko Nango ◽  
Kensuke Tono ◽  
Tetsunari Kimura ◽  
Shigeki Owada ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Free Electron ◽  
Good Choice ◽  
Cubic Phase ◽  
Liquid Droplets ◽  
Optical Pump ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

scholarly journals Rapid sample delivery for megahertz serial crystallography at X-ray FELs

IUCrJ ◽  
2018 ◽  
Vol 5 (5) ◽  
pp. 574-584 ◽  
Author(s):  
Max O. Wiedorn ◽  
Salah Awel ◽  
Andrew J. Morgan ◽  
Kartik Ayyer ◽  
Yaroslav Gevorkov ◽  
...  
Keyword(s):  
Bragg Diffraction ◽  
X Rays ◽  
Free Electron Lasers ◽  
X Ray ◽  
Damaged Material ◽  
Optical Images ◽  
Subsequent Measurement ◽  
Serial Crystallography ◽  
Liquid Microjets ◽  
Serial Femtosecond Crystallography

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s−1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.

scholarly journals Advances in Diffraction Studies of Light-Induced Transient Species in Molecular Crystals and Selected Complementary Techniques

Crystals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1345
Author(s):  
Krystyna A. Deresz ◽  
Piotr Łaski ◽  
Radosław Kamiński ◽  
Katarzyna N. Jarzembska
Keyword(s):  
Free Electron Lasers ◽  
Transient Species ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Recent Developments ◽  
Complementary Techniques ◽  
X Ray Absorption ◽  
Serial Femtosecond Crystallography ◽  
Major Emphasis

The review provides a summary of the current methods of tracing photoexcitation processes and structural dynamics in the solid state, putting major emphasis on the X-ray diffraction techniques (time-resolved Laue diffraction on synchrotron sources and time-resolved serial femtosecond crystallography on X-ray free-electron lasers). The recent developments and nowadays experimental possibilities in the field are discussed along with the data processing and analysis approaches, and illustrated with some striking literature examples of the respective successful studies. Selected complementary methods, such as ultrafast electron diffraction or time-resolved X-ray absorption spectroscopy, are briefly presented.

scholarly journals First Experiments in Structural Biology at the European X-ray Free-Electron Laser

2020 ◽  
Vol 10 (10) ◽  
pp. 3642 ◽  
Author(s):  
Grant Mills ◽  
Richard Bean ◽  
Adrian P. Mancuso
Keyword(s):  
Structural Biology ◽  
Structure Determination ◽  
Free Electron ◽  
Free Electron Laser ◽  
Pulse Frequency ◽  
Free Electron Lasers ◽  
Time Resolved ◽  
New Era ◽  
X Ray ◽  
Rapid Pulse

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

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