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Featured Application: Recently upgraded inject-and-transfer system will improve beamtime efficiency for serial femtosecond crystallography. Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) provides room‐temperature crystal structures with minimal radiation damage and facilitates the comprehension of molecular dynamics through time‐resolved studies. In SX experiments, it is important to deliver a large number of crystal samples to the X‐ray interaction point in a serial and stable manner. The advantage of crystal delivery in a viscous medium via a capillary is the ability to deliver all of the crystal samples to the X‐ray interaction point at a low flow rate; however, the capillary often breaks during handling and high X‐ray absorption can occur at low energy states. This study aimed to develop a stable system for sample delivery in a viscous medium via a polyimide‐based single‐channel microfluidic (PSM) chip for SX. Since this microfluidic chip comprises a polyimide film, it has high tensile strength and higher X‐ray transmittance than a quartz capillary. The PSM chip was connected to a syringe containing the microcrystals embedded in viscous medium. The channel of the PSM chip was aligned to the X‐ray path, and the viscous medium containing lysozyme crystals was stably delivered using a syringe pump at a flow rate of 100 nl min−1. Room‐temperature lysozyme crystal structures were successfully determined at 1.85 Å resolution. This method would greatly facilitate sample delivery for SX experiments using synchrotron X‐rays. [ABSTRACT FROM AUTHOR]

The serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments. [ABSTRACT FROM AUTHOR]

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 femtosecond crystallography (SFX) enables the determination of the room‐temperature crystal structure of macromolecules, as well as providing time‐resolved molecular dynamics data in pump–probe experiments. Fixed‐target SFX (FT‐SFX) can minimize sample consumption and physical effects on crystals during sample delivery. In FT‐SFX studies, having a sample holder that can stably fix crystal samples is one of the key elements required for efficient data collection. Hence, development of sample holders from new materials capable of supporting various crystal sizes and shapes may expand the applications of FT‐SFX. Here, a viscous‐media‐based crystal support in a sample holder for FT‐SFX is introduced. Crystal samples were embedded in viscous media, namely gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, 10–15%(w/v) viscous gelatin and 1–4%(w/v) agarose gel stably supported crystals between two polyimide films, thereby preventing the crystals from descending owing to gravity. Using this method, FT‐SFX experiments were performed with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room‐temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. The glucose isomerase and lysozyme diffraction analyses were not impeded by excessive background scattering from the viscous media. This method is useful for delivering crystal samples of various sizes and shapes in FT‐SFX experiments. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of the 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, drastically reducing sample consumption. In serial millisecond crystallography using a viscous medium via a capillary, crystals of lysozyme embedded in agarose, which produce an unstable injection stream at atmospheric pressure, and crystals of glucose isomerase embedded in gelatin, which is known to be problematic for open‐extruder operation, were stably delivered at a flow rate of 100 nl min−1. The room‐temperature crystal structures of lysozyme and glucose isomerase were successfully determined at 1.85 and 1.70 Å resolutions, respectively. This simple but highly efficient sample‐delivery method can allow researchers to deliver crystals precisely to an X‐ray beam in SX experiments. [ABSTRACT FROM AUTHOR]

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) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment. [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]