Experimental approaches for time-resolved serial femtosecond crystallography at PAL-XFEL

Understanding the structures and dynamics of biomolecules and chemical compounds is crucial for deciphering their molecular functions and mechanisms. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) is a useful technique for determining structures at room temperature, while minimizing radiation damage. Time-resolved serial femtosecond crystallography (TR-SFX), which uses an optical laser or a mixing device, allows molecular dynamic visualization during a reaction at specific time points. Because the XFEL beamline has unique properties for beams and instruments, understanding the beamline system is essential to conduct TR-SFX experiments and develop related technologies. In this study, we introduce an experimental system for performing TR-SFX using a Nano Crystallography and Coherent Imaging (NCI) experimental hutch at the Pohang Accelerator Laboratory XFEL (PAL-XFEL). Specifically, we present the XFEL properties of the PAL-XFEL and the main instruments in the NCI experimental hutch. In addition, the characteristics and uses of the sample delivery methods for TR-SFX and the general sample preparation process are discussed. Furthermore, the general time schedule and experimental procedures for TR-SFX during the beam time are outlined, along with data analysis programs. This chapter contributes to understanding the performance of TR-SFX experiments conducted at the PAL-XFEL NCI experimental hutch.