Effect of Crystal-to-Detector Distance Variations on Serial Femtosecond Crystallography Data Collected at PAL-XFEL

Serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) enables the determination of room-temperature structures of biological macromolecules without radiation damage. The accuracy of detector geometry parameters, including the crystal-to-detector distance (CTDD), is critical for reliable data processing. In SFX experiments, the CTDD may shift during data collection due to changes in the experimental setup or installation of the sample delivery system. Such CTDD variations can affect the quality of SFX datasets; however, their impact has not been fully elucidated in the context of SFX data processing. In this study, we investigated the influence of CTDD variations on SFX datasets collected at Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) with thermolysin, lysozyme, and glucose isomerase crystals processed by four indexing algorithms. At the optimized CTDD, the distribution of unit cell parameters exhibited a Gaussian pattern; however, it became distorted as the CTDD deviated further from the optimal value. Data analysis indicated that the CTDD tolerance for successful data processing and structure determination was approximately ±3–5 mm from the optimized CTDD. These findings provide insight into indexing behavior in SFX data processing at PAL-XFEL and offer practical guidance for improving data processing efficiency.