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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]

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) 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]

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]