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 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 A new approach to time-resolved X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1434-C1434
Author(s):  
Briony Yorke ◽  
Arwen Pearson ◽  
Godfrey Beddard ◽  
Robin Owen
Keyword(s):  
Molecular Mechanisms ◽  
Structural Information ◽  
Information Storage ◽  
High Time Resolution ◽  
Time Resolved ◽  
New Approach ◽  
X Ray ◽  
X Ray Crystallography ◽  
Current State ◽  
Initial Results

Time-resolved crystallography is able to provide four-dimensional structural information about short-lived intermediate states, with near-atomic resolution. This information can be used to elucidate molecular mechanisms relevant to areas such as drug-design, chemical and biological sensors, and energy and information storage. The current state of the art time-resolved experiments can reach picosecond time-resolutions using Laue crystallography but such experiments can only be carried out at a few beamlines worldwide.We have developed a new transform time-resolved method that can be performed using a monochromatic beamline at a synchrotron and still achieve high time-resolution, vastly increasing the accessibility of such experiments. Here we present initial results demonstrating the method.

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 Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 306-323 ◽  
Author(s):  
Alexander M. Wolff ◽  
Iris D. Young ◽  
Raymond G. Sierra ◽  
Aaron S. Brewster ◽  
Michael W. Martynowycz ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Structural Information ◽  
Biological Macromolecules ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Delivery Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Physical Conditions ◽  
Serial Crystallography

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

scholarly journals Allosteric Regulation of the EphA2 Receptor Intracellular Region by Serine/Threonine Kinases

2021 ◽  
Author(s):  
Bernhard C. Lechtenberg ◽  
Marina P. Gehring ◽  
Taylor P. Light ◽  
Mike W. Matsumoto ◽  
Kalina Hristova ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Cell Communication ◽  
Kinase Domain ◽  
Eph Receptor ◽  
X Ray ◽  
X Ray Crystallography ◽  
Exchange Mass Spectrometry ◽  
Intracellular Region

ABSTRACTEph receptor tyrosine kinases play a key role in cell-cell communication. However, lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered detailed understanding of their signaling mechanisms. Here, we use an integrative structural biology approach combining X-ray crystallography, small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry, to gain the first insights into the structure and dynamics of the entire EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling that depends on serine/threonine phosphorylation of the linker connecting the EphA2 kinase and SAM domains. We uncovered two distinct molecular mechanisms that may function in concert to mediate the effects of linker phosphorylation through an orchestrated allosteric regulatory network. The first involves a shift in the equilibrium between a “closed” configuration of the EphA2 intracellular region and an “open” more extended configuration induced by the accumulation of phosphorylation sites in the linker. This implies that cooperation of multiple serine/threonine kinase signaling networks is necessary to promote robust EphA2 non-canonical signaling. The second involves allosteric rearrangements in the kinase domain and juxtamembrane segment induced by phosphorylation of some linker residues, suggesting a link between EphA2 non-canonical signaling and canonical signaling through tyrosine phosphorylation. Given the key role of EphA2 in cancer malignancy, this new knowledge can inform therapeutic strategies.

scholarly journals Sample delivery using capillary and delivery medium for serial crystallography

2019 ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Capillary Tube ◽  
Low Flow ◽  
Innovative Technology ◽  
X Rays ◽  
Delivery Method ◽  
Interaction Point ◽  
Crystal Sample ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

AbstractSerial crystallography (SX) is an innovative technology in structural biology that enables the visualization of molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques and drastically reducing sample consumption. This simple but highly efficient sample delivery method can allow researchers to deliver crystals precisely to X-rays in SX experiments.

scholarly journals Lard Injection Matrix for Serial Crystallography

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.

scholarly journals Shortening injection matrix for serial crystallography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ki Hyun Nam
Keyword(s):  
Crystal Structures ◽  
Room Temperature ◽  
Low Cost ◽  
Glucose Isomerase ◽  
Low Flow ◽  
Cubic Phase ◽  
Interaction Point ◽  
X Ray ◽  
Serial Crystallography ◽  
Delivery Medium

AbstractSerial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

scholarly journals CrystFEL: a software suite for snapshot serial crystallography

2012 ◽  
Vol 45 (2) ◽  
pp. 335-341 ◽  
Author(s):  
Thomas A. White ◽  
Richard A. Kirian ◽  
Andrew V. Martin ◽  
Andrew Aquila ◽  
Karol Nass ◽  
...  
Keyword(s):  
Structural Information ◽  
Software Suite ◽  
X Ray ◽  
Large Numbers ◽  
Small Crystals ◽  
Serial Crystallography ◽  
Diffraction Patterns ◽  
Complicated Geometries ◽  
Serial Femtosecond Crystallography

In order to address the specific needs of the emerging technique of `serial femtosecond crystallography', in which structural information is obtained from small crystals illuminated by an X-ray free-electron laser, a new software suite has been created. The constituent programs deal with viewing, indexing, integrating, merging and evaluating the quality of the data, and also simulating patterns. The specific challenges addressed chiefly concern the indexing and integration of large numbers of diffraction patterns in an automated manner, and so the software is designed to be fast and to make use of multi-core hardware. Other constituent programs deal with the merging and scaling of large numbers of intensities from randomly oriented snapshot diffraction patterns. The suite uses a generalized representation of a detector to ease the use of more complicated geometries than those familiar in conventional crystallography. The suite is written in C with supporting Perl and shell scripts, and is available as source code under version 3 or later of the GNU General Public License.

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.

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