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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 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]