scholarly journals Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

10.3791/59087 ◽  
2019 ◽  
Author(s):  
Daniel James ◽  
Tobias Weinert ◽  
Petr Skopintsev ◽  
Antonia Furrer ◽  
Dardan Gashi ◽  
...  
Keyword(s):  
High Viscosity ◽  
Time Resolved ◽  
X Ray ◽  
Serial Femtosecond Crystallography

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

2019 ◽  
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.

scholarly journals Viscous hydrophilic injection matrices for serial crystallography

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 400-410 ◽  
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.

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

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 412-425 ◽  
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.

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

2019 ◽  
Vol 9 (24) ◽  
pp. 5505 ◽  
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.

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

2019 ◽  
Vol 75 (2) ◽  
pp. 178-191 ◽  
Author(s):  
Marie Luise Grünbein ◽  
Gabriela Nass Kovacs
Keyword(s):  
Data Collection ◽  
Free Electron ◽  
High Viscosity ◽  
Free Electron Lasers ◽  
Time Resolved ◽  
Liquid Injection ◽  
Serial Data ◽  
Current Sample ◽  
Serial Femtosecond Crystallography ◽  
Delivery Techniques

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.

scholarly journals Serial Femtosecond Crystallography user's consortium apparatus at European XFEL

2014 ◽  
Vol 70 (a1) ◽  
pp. C1748-C1748
Author(s):  
Marc Messerschmidt ◽  
Leonard Chavas ◽  
Sunil Ananthaneni ◽  
Hamidreza Dadgostar ◽  
Heinz Graafsma ◽  
...  
Keyword(s):  
Protein Crystal ◽  
Pixel Detector ◽  
Single Particles ◽  
Time Resolved ◽  
X Ray ◽  
Overall Design ◽  
Laser Facility ◽  
Common Problems ◽  
Automated Procedures ◽  
Serial Femtosecond Crystallography

The Serial Femtosecond Crystallography (SFX) user's consortium apparatus is to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument of the European X-ray Free-Electron Laser facility (XFEL.EU) [1, 2]. The XFEL.EU will provide ultra-short, highly intense, coherent X-ray pulses at an unprecedented repetition rate. The experimental setup and methodological approaches of many scientific areas will be transformed, including structural biology that could potentially overcome common problems and bottlenecks encountered in crystallography, such as creating large crystals, dealing with radiation damage, or understanding sub-picosecond time-resolved phenomena. The key concept of the SFX method is based on the kinetic insertion of protein crystal samples in solution via a gas dynamic virtual nozzle jet and recording diffraction signals of individual, randomly oriented crystals passing through the XFEL beam, as first demonstrated by Chapman et al. [3]. The SFX-apparatus will refocus the beam spent by the SPB instrument into a second interaction region, in some cases enabling two parallel experiments. The planned photon energy range at the SPB instrument is from 3 to 16 keV. The Adaptive Gain Integrating Pixel Detector (AGIPD) is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX-apparatus. The AGIPD is designed to store over 350 data frames from successive pulses, and aims to collect more than 3,000 images per second. Together with the implementation of automated procedures for sample exchange and injection, high-throughput nanocrystallography experiments can be integrated at the SFX-apparatus. In this work, we review the overall design of the SFX-apparatus and discuss the main parameters and challenges

Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments

2017 ◽  
Vol 50 (3) ◽  
pp. 932-939 ◽  
Author(s):  
Giorgio Schirò ◽  
Joyce Woodhouse ◽  
Martin Weik ◽  
Ilme Schlichting ◽  
Robert L. Shoeman
Keyword(s):  
Structural Changes ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Super Resolution ◽  
High Viscosity ◽  
Optical Pump ◽  
Time Resolved ◽  
Efficient System ◽  
X Ray ◽  
Photosensitive Proteins

Proteins that change their structure in response to light absorption regulate many functional processes in living cells. Moreover, biotechnological approaches like optogenetics and super-resolution fluorescence microscopy recently triggered the generation of new genetically modified photosensitive proteins. Light-induced structural changes in photosensitive proteins can be studied by time-resolved serial femtosecond crystallography (SFX), an X-ray diffraction technique that allows the determination of macromolecular structures at X-ray free-electron lasers from a large number of nano- to micro-sized crystals. This article describes a simple and efficient system for converting photosensitive proteins into light-induced semi-stationary states by inline laser illumination prior to sample injection with a gas-focused liquid jet and subsequent optical pump–X-ray probe exposure. The simple setup of this device makes it suitable for integration into other liquid injectors (like electro-spinning and electro-kinetic injectors) and potentially also in high-viscosity extruders, provided that embedding microcrystals in viscous media does not alter protein photophysical properties. The functioning of the device is demonstrated with an example of a photoswitchable fluorescent protein pre-illuminated (photoactivated) for time-resolved SFX experiments. The device can be easily adapted for the conversion in time-resolved SFX experiments of other microcrystalline proteins, such as photosystems, phytochromes and rhodopsins.

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