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Serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) enables the determination of room-temperature structures without causing radiation damage. Using an optical pump-probe or mix-and-injection, SFX enables the intermediate state visualization of a molecular reaction. In SFX experiments, serial and stable microcrystal delivery to the X-ray interaction point is vital for reasonable data collection and efficient beam time. The Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) facility established SFX instruments at a nanocrystallography and coherent imaging (NCI) experimental station. Various sample delivery methods, including injection, fixed-target scanning, and hybrid methods, have been developed and applied to collect XFEL diffraction data. Herein, we report the currently available sample delivery methods for SFX at the NCI experimental station at the PAL-XFEL. This article will help PAL-XFEL users access the SFX system for their experiments. [ABSTRACT FROM AUTHOR]

Featured Application: Our results provide insight into the material selection and directions to pursue in developing sample delivery devices used in XFEL studies. A crystal delivery system is essential in serial femtosecond crystallography experiments using an X-ray free-electron laser (XFEL). Investigating the XFEL-induced radiation damage to materials potentially applicable to sample delivery devices is vital for developing a sample delivery system. In this study, we investigated the radiation damage caused by an XFEL to polydimethylsiloxane (PDMS) and polyimide (PI), which are widely used as sample delivery materials in synchrotron X-rays. Upon XFEL exposure, the PDMS film produced irregularly shaped and sized holes, whereas the PI film produced relatively regular shaped and sized holes. When XFELs were exposed to the channel of the PDMS-based microfluidic device, holes were generated on the film by the radiation damage and the microfluidic device and the internal channel region were structurally destroyed. The PI-based microfluidic device experienced no structural destruction, except for the holes generated by the XFEL. However, as the XFELs were continuously exposed, bubbles generated from the solution due to radiation damage; the accumulation of these bubbles interfered with the path of the inner channel of the microfluidic device. Our results will not only help understand the phenomenon of radiation damage of PDMS and PI films by XFEL, but also provide insight into the directions to pursue in developing applications of PDMS and PI films in XFEL studies. [ABSTRACT FROM AUTHOR]

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X‐ray beam position in a stable manner. Using this system, the room‐temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Å and 1.75 Å, respectively, were determined. This straightforward syringe pump‐based sample delivery system can be utilized in SX. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9–2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]