## 1 Introduction  
X-ray crystallography is a powerful tool for determining the protein structure at high spatial resolution. To date, atomic structural information has been valuable in elucidating the mechanisms and designing pharmaceuticals, because of the close relation between the tertiary structure of proteins and their functions. In general, the tertiary structure of proteins at work changes from the initial structure before a reaction; however, it remains a challenge that conventional X-ray crystallography captures protein dynamic structures. The Laue method with polychromatic synchrotron X-rays [1] or cryotrapping to capture intermediates at cryogenic temperatures has so far been used to observe the active state in which a protein functions. Nevertheless, there is a limit for which samples can be applied to the Laue method due to technical difficulties. For instance, there is a tendency for crystals to suffer from radiation damage that is caused by X-rays from synchrotron radiation sources, which results in artificial structural changes in proteins. Moreover, protein reactions may proceed, even at cryogenic temperatures, and cause changes that vary from time-dependent changes upon a reaction initiator, such as photoexcitation. Indeed, the determination of protein dynamic structures at atomic resolution has been one of the hefty challenges in structural biology for quite a long time. X-ray free-electron lasers (XFELs) produce intense, ultrashort pulses; as such, XFELs have enabled room-temperature protein structure determination via diffraction patterns from microcrystals before any manifestation of radiation damage [2]. Furthermore, these femtosecond X-ray pulses from XFEL can capture rapid structural changes in proteins to allow for the visualization of the dynamic process in proteins at atomic resolution. Hence, serial femtosecond crystallography (SFX) is a recently established method for protein structure determination by XFEL [3; 4]. In SFX, the microcrystals are continuously delivered into an intersection point with an XFEL beam, because the preceding XFEL pulse destroys the samples. Subsequently, intense X-ray pulses afford still diffraction patterns from randomly oriented microcrystals, which results in a protein tertiary structure in a condition close to physiological temperature. In Japan, the SPring-8 Angstrom Compact Free-Electron Laser (SACLA) started user operations in 2012 [5] after the Linac Coherent Light Source (LCLS) in the USA [6]. A consortium of researchers from structure biology, physics, engineering, and XFEL science was organized in 2012 for an SFX project named \"Rapid Structure Determination System for Drug-target Proteins Using the X-ray Free Electron Laser\" that was directed by Prof. [PERSON] under the X-ray Free-Electron Laser Priority Strategy Program of the Ministry of Education, Culture, Sports, Science & Technology in Japan (MEXT). The project established the foundation of devices and techniques for SFX at SACLA for the first five years. This foundation has also led to the development of a setup for pump-probe time-resolved serial femtosecond crystallography (TR-SFX), which uses a pump laser pulse to trigger reactions. In 2015, the first experiment of pump-probe TR-SFX at SACLA was conducted while using a light-driven proton pump, bacteriorhodopsin (bR), and, in early 2016, we succeeded in observing the structural changes in bR during proton transfer at thirteen-time points from nanoseconds to milliseconds subsequent to light excitation while using a pump laser [7]. The result was the first three-dimensional movie of large structural changes throughout a protein, which reveals how such structural changes in bR achieve unidirectional proton transport across the membrane. Pump-probe TR-SFX techniques have been further applied to various proteins, including photosystem II (PSII), which is a huge membrane-protein complex that catalyzes light-driven water oxidation at its catalytic center [8].