Preliminary Serial Femtosecond Crystallography Studies of Myoglobin from Equine Skeletal Muscle

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.