scholarly journals An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Agata Butryn ◽  
Philipp S. Simon ◽  
Pierre Aller ◽  
Philip Hinchliffe ◽  
Ramzi N. Massad ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Time Resolved ◽  
X Ray ◽  
Drop Method ◽  
Inducible Systems ◽  
Serial Crystallography ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Ligand Diffusion ◽  
Serial Femtosecond Crystallography

AbstractSerial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

scholarly journals Sample Delivery Media for Serial Crystallography

2019 ◽  
Vol 20 (5) ◽  
pp. 1094 ◽  
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.

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

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.

scholarly journals Serial X-ray Crystallography

Crystals ◽  
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.

scholarly journals The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation

2019 ◽  
Vol 26 (3) ◽  
pp. 660-676 ◽  
Author(s):  
Adrian P. Mancuso ◽  
Andrew Aquila ◽  
Lewis Batchelor ◽  
Richard J. Bean ◽  
Johan Bielecki ◽  
...  
Keyword(s):  
Structure Determination ◽  
High Repetition Rate ◽  
Optical Systems ◽  
Diagnostic Tools ◽  
Imaging Method ◽  
Single Particles ◽  
X Ray ◽  
Data Rates ◽  
Serial Crystallography ◽  
Serial Femtosecond Crystallography

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation – in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.

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 Approach of Serial Crystallography

Crystals ◽  
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.

scholarly journals Serial crystallography using automated drop dispensing

2021 ◽  
Vol 28 (5) ◽  
Author(s):  
Zhen Su ◽  
Joshua Cantlon ◽  
Lacey Douthit ◽  
Max Wiedorn ◽  
Sébastien Boutet ◽  
...  
Keyword(s):  
Free Electron Laser ◽  
Protein Crystal ◽  
Cross Contamination ◽  
X Ray ◽  
On Demand ◽  
Drop On Demand ◽  
Serial Crystallography ◽  
Cycling System ◽  
Serial Femtosecond Crystallography

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

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.

Sign in / Sign up

Export Citation Format

Share Document