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Proteins can form crystals spontaneously without crystallization experiments. These crystals can be used to determine three-dimensional structures. However, when X-ray diffraction is poor, crystal optimization is required to obtain a high-resolution crystal structure. Endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) spontaneously formed microcrystals after affinity purification and concentration; however, most HviGH11 microcrystals showed poor diffraction in the synchrotron X-ray and X-ray free-electron laser, so a complete three-dimensional structure could not be obtained. This study presents a method to improve the crystal quality of spontaneously grown HviGH11 microcrystals. The crystallization screening results revealed that temperature, pH, and salt were not crucial factors in increasing the solubility or preventing the spontaneous crystal growth of HviGH11. Conversely, the addition of polyethylene glycols (PEGs) as a precipitant facilitated the growth of larger HviGH11 crystals. The improved large HviGH11 crystal showed a diffraction of up to 1.95 Å when exposed to synchrotron X-rays, providing a complete three-dimensional structural dataset. Based on the nucleation rate equation, it was suggested that PEG increases the viscosity of the protein solution rather than promoting nucleation. This increase in viscosity reduced nucleation and facilitated the growth of larger HviGH11 crystals. These results provide valuable insights for future experiments aimed at increasing the size of spontaneously grown crystals. [ABSTRACT FROM AUTHOR]

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]

When X-rays pass through a material, radiation damage occurs, and heat is generated at the X-ray interaction point, which can then be transferred around the X-ray irradiation site. This X-ray-induced heat transfer can affect the temperature of the sample and consequently the experimental environment in serial crystallography (SX) experiments. Here, we investigated radiation damage and measured the level of heating in the vicinity of the X-ray interaction point. In our experimental setup, when water, crystallization solution, and crystal suspension in a glass tube were exposed to X-rays, a temperature increase of approximately 1.0 °C occurred in the vicinity of the X-ray interaction point, with the heat generated by both the sample and the capillary. When Cu and Al/Zn plates were exposed to X-rays, the temperature around the X-ray exposure point increased by approximately 0.3 and 0.4 °C, respectively. The range of temperature rise decreased as the distance from the X-ray exposure point on the Al plate increased. The heat generated by the X-rays and the rise of the heat could be reduced by discontinuously transmitting the X-rays using the shutter. Our results provide useful information for obtaining more accurate experimental parameters. [ABSTRACT FROM AUTHOR]

Traditional macromolecular crystallography (MX) and recently spotlighted cryogenic electron microscopy (Cryo-EM) techniques have contributed greatly to the development of macromolecule structures and the related fields [[1], [3], [5], [7]]. Starting with serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) [[11]], the SX technique is widely applied to serial synchrotron crystallography (SSX) using synchrotron X-rays [[16]], which can aid in obtaining in-depth and accurate structural information. Kim and Nam reported experiments on serial crystallography using pink-beam X-rays in PLS-II [[25]]. 10.3390/cryst12020138 24 Nam K.H. Processing of Multicrystal Diffraction Patterns in Macromolecular Crystallography Using Serial Crystallography Programs. [Extracted from the article]

Serial crystallography (SX) using X-ray free-electron lasers or synchrotron X-rays is an emerging technique for determining the room-temperature structure of macro- and small-molecules [[9]]. Molecular dynamics are observed in all molecules, from small molecules to macromolecules, and provide information about their intrinsic structure in a spatiotemporal context within a specific environment. All natural molecules have their own physical, chemical, or biological properties and functions. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) is an emerging X-ray crystallographic method for determining macromolecule structures. It can address concerns regarding the limitations of data collected by conventional crystallography techniques, which require cryogenic-temperature environments and allow crystals to accumulate radiation damage. Time-resolved SX studies using the pump-probe methodology provide useful information for understanding macromolecular mechanisms and structure fluctuation dynamics. This Special Issue deals with the serial crystallography approach using an X-ray free electron laser (XFEL) and synchrotron X-ray source, and reviews recent SX research involving synchrotron use. These reports provide insights into future serial crystallography research trends and approaches. [ABSTRACT FROM AUTHOR]

The ultrabright and ultrashort pulses produced at X-ray free electron lasers (XFELs) has enabled studies of crystallized molecular machines at work under 'native' conditions at room temperature by the so-called time-resolved serial femtosecond crystallography (TR-SFX) technique. Since early TR-SFX experiments were conducted at XFELs, it has been largely reported in the literature that time-resolved X-ray experiments at synchrotrons are no longer feasible or are impractical due to the severe technical limitations of these radiation sources. The transfer of the serial crystallography approach to newest synchrotrons upgraded for higher flux density and with beamlines using sophisticated focusing optics, submicron beam diameters and fast low-noise photon-counting detectors offers a way to overcome these difficulties opening new and exciting possibilities. In fact, there is an increasing amount of publications reporting new findings in structural dynamics of protein macromolecules by using time resolved crystallography from microcrystals at synchrotron sources. This review gathers information to provide an overview of the recent work and the advances made in this filed in the past years, as well as outlines future perspectives at the next generation of synchrotron sources and the upcoming compact pulsed X-ray sources. [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]