Microcrystal delivery methods are pivotal in the use of serial femtosecond crystallography (SFX) to resolve the macromolecular structures of proteins. Here, the development of a novel technique and instruments for efficiently delivering microcrystals for SFX are presented. The new method, which relies on a one-dimensional fixed-target system that includes a microcrystal container, consumes an extremely low amount of sample compared with conventional two-dimensional fixed-target techniques at ambient temperature. This novel system can deliver soluble microcrystals without highly viscous carrier media and, moreover, can be used as a microcrystal growth device for SFX. Diffraction data collection utilizing this advanced technique along with a real-time visual servo scan system has been successfully demonstrated for the structure determination of proteinase K microcrystals at 1.85 Å resolution.

The X-ray free-electron laser of the Pohang Accelerator Laboratory (PAL-XFEL) was opened to users in 2017. Since then, significant progress has been made in PAL-XFEL operation and beamline experiments. This includes increasing the FEL pulse energy, increasing the FEL photon energy, generating self-seeding FEL, and trials of two-color operation. In the beamline, new instruments or endstations have been added or are being prepared. Overall, beamline operation has been stabilized since its initiation, which has enabled excellent scientific results through efficient user experiments. In this paper, we describe details of the recent progress of the PAL-XFEL.

HIV-1 integrase (HIV-1 IN) is an enzyme produced by the HIV-1 virus that integrates genetic material of the virus into the DNA of infected human cells. HIV-1 IN acts as a key component of the Retroviral Pre-Integration Complex (PIC). Protein dynamics could play an important role during the catalysis of HIV-1 IN; however, this process has not yet been fully elucidated. X-ray free electron laser (XFEL) together with nuclear magnetic resonance (NMR) could provide information regarding the dynamics during this catalysis reaction. Here, we report the non-cryogenic crystal structure of HIV-1 IN catalytic core domain at 2.5 Å using microcrystals in XFELs. Compared to the cryogenic structure at 2.1 Å using conventional synchrotron crystallography, there was a good agreement between the two structures, except for a catalytic triad formed by Asp64, Asp116, and Glu152 (DDE) and the lens epithelium-derived growth factor binding sites. The helix III region of the 140–153 residues near the active site and the DDE triad show a higher dynamic profile in the non-cryogenic structure, which is comparable to dynamics data obtained from NMR spectroscopy in solution state.

Myoglobin (Mb), a heme-containing protein, plays crucial roles in storing and transporting oxygen in muscle cells. Various Mb structures have been extensively determined using conventional cryogenic crystallography, providing valuable information for understanding the molecular mechanisms of the protein. However, this approach has limitations attributable to cryogenic temperatures and radiation damage. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers is an emerging technique that enables the determination of biologically relevant room-temperature structures without causing radiation damage. In this study, we assessed the crystallization, collection, and processing of SFX diffraction data of Mb from equine skeletal muscle. Needle- and needle cluster-shaped Mb crystals were obtained using the microbatch method. Fixed-target SFX data collection was performed at the Pohang Accelerator Laboratory X-ray Free Electron Laser, yielding 1389 indexed diffraction patterns. The phase problem was solved by molecular replacement. The preliminary Mb structure determined at 2.3-Å resolution in this study exhibited subtle structural differences in the heme environment compared with previously reported Mb structures determined by SFX. These results both confirm the feasibility of myoglobin SFX experiments and establish a foundation for future time-resolved studies aiming to visualize ligand binding and oxygen transport.

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.