LAUSR

Serial crystallography (SX) using X-ray free-electron lasers (XFEL) and synchrotron X-rays is an emerging X-ray crystallography technique to determine the structure of macromolecules at room temperature or near-physiological temperature with minimal radiation damage (Chapman et al., 2011; Boutet et al., 2012; Chapman et al., 2014; Stellato et al., 2014; Johansson et al., 2017; Standfuss and Spence, 2017; Nam, 2019; Nam, 2021b; Durdagi et al., 2021; Nam, 2022c). This technique is used for studying time-resolved molecular mechanisms through pump-and-probe experiments with an optical laser or a liquid application (e.g., substrate or inhibitors) (Spence, 2014; Schulz et al., 2018; Schmidt, 2019; Butryn et al., 2021; Martin-Garcia, 2021). The SX technique overcomes the experimental limitations of traditional X-ray crystallography. This technique causes minimal radiation damage, does not need a cryogenic environment, and provides dynamic structural information; furthermore, it provides biologically relevant structural information with accurate visuals depicting the molecular mechanism (Chapman et al., 2011; Boutet et al., 2012; Chapman et al., 2014; Schmidt, 2019; Orville, 2020; Pearson and Mehrabi, 2020; Nam, 2021a; Nam, 2022c). In an SX experiment, a large number of crystals are serially delivered to an X-ray interaction point via various sample delivery techniques, such as injectors injector (DePonte et al., 2008; Weierstall et al., 2014), syringes with viscous medium (Sugahara et al., 2015; Park and Nam, 2019; Nam, 2020a; Nam, 2022a), fixed-target scanning (Hunter et al., 2014; Murray et al., 2015; Lee et al., 2019; Lee et al., 2020; Park et al., 2020; Nam et al., 2021), capillaries (Stellato et al., 2014; Nam, 2020b), convey belts (Beyerlein et al., 2017a), and microfluidics (Knoska et al., 2020; Monteiro et al., 2020; Nam and Cho, 2021). Crystals are exposed to X-rays only once for a short period of time at the XFEL (fs level) or synchrotron (ms level). A large number of images (ranging from thousands to millions) are collected to determine the three-dimensional structure of macromolecules during SX data collection (Schmidt, 2019). Delivering the crystals spatiotemporally in a continuous manner at the X-ray interaction location during SX data collection is experimentally impossible. Hence, the collected data include images that contain diffraction information generated while penetrating X-ray crystals and other images that do not penetrate the crystal. In general, four types of images can be collected, as follows: 1) single crystal diffraction, 2) multicrystal diffraction, 3) unwanted diffraction or scattering (salt or crystal delivery materials), and 4) diffraction-free images (Figure 1A). In SX technology, a “hit” denotes a diffraction pattern with the minimum number of detectable Bragg peaks (Barty et al., 2014). As only hit images containing Bragg peaks are needed for structure determination, hit images are filtered from whole images using image filtering programs and employed for the next data processing step. Filtering the hit image has the following two advantages: 1) Filtering only hit images reduces the time needed for the next data processing step and aids in the efficient utilization of available computing resources. 2) Excluding the non-hit images reduces storage consumption and file conversion time (e.g., cxi to hdf5). Meanwhile, the hit rate (ratio) is Edited by: Anthony Karl Mittermaier, McGill University, Canada