Structural insights into the ADP-bound p97/VCP ND1 hexamer revealed by room-temperature XFEL crystallography

p97/VCP ATPase uses adenosine triphosphate (ATP) hydrolysis to remodel protein assemblies and unfold substrates across diverse cellular processes, including protein quality control, membrane fusion, and chromatin regulation. Although cryogenic structural studies have provided valuable snapshots of distinct nucleotide states, they often suppress the conformational variations that are intrinsic to the protein function. Here, we present a comparative structural analysis of the ADP-bound human p97 ND1 hexamer using room-temperature X-ray free-electron laser (XFEL) and cryogenic synchrotron crystallography. While the fundamental hexameric architecture is conserved across both conditions, the room-temperature XFEL structure reveals significantly enhanced conformational variability. Notably, the N-terminal domain exhibits increased flexibility and positional variations. Furthermore, the room-temperature structure reveals an extended, ordered active-site water network that connects the nucleotide-binding pocket to the bulk solvent, a feature absent under cryogenic conditions. Interestingly, the ADP ribose undergoes a pucker transition from a canonical C2'endo to a C3'-endo conformation, revealing altered active-site conformations. Together, these structures highlight the intrinsic conformational flexibility of the p97 ND1 fragment using room-temperature XFEL crystallography as a powerful approach for identifying temperature-dependent structural flexibility of ATPases.