Temperature-Dependent Structural Dynamics of SOD1 Revealed by Serial Synchrotron Crystallography

Objective: To determine the high-resolution structure of human Copper-Zinc superoxide dismutase (hSOD1), an antioxidant enzyme whose mutations cause amyotrophic lateral sclerosis (ALS), under near-physiological conditions. Because SOD1 is intrinsically dynamic, capturing its structure at ambient temperature is key to understanding how temperature modulates its conformational flexibility, ensemble and functional states relevant to both catalysis and disease.Materials and Methods: Recombinant hSOD1 was expressed in E. coli, purified by affinity and size-exclusion chromatography, and crystallized at ambient temperature. Serial synchrotron crystallography (SSX) data were collected at 293 K at the EMBL P14-2 Time-Resolved Experiments with Crystallography (T-REXX) beamline at PETRA III, and compared with a 100 K cryogenic at the Diamond Light Source beamline (I03). Both datasets were processed and refined using CCP4 suite and PHENIX packages. B-factor distributions, per-residue RMSD values, and conformational differences were analyzed to quantify temperature-dependent effects.Results: The ambient-temperature SOD1SSX structure was determined at 2.3 Å resolution (PDB ID:9XJ0 this work) and closely matched its 2.37 Å cryogenic counterpart (SOD1CRYO, PDB ID:9XJI this work), both obtained from identical crystallization conditions in the hexagonal P6₃ space group. Cryocooling caused a 3.8% contraction in unit-cell volume, consistent with lattice densification and a 5.2% reduction in molecular surface volume. Despite the overall similarities, the ambient-temperature model revealed localized conformational differences in solvent-exposed loop residues, particularly Ser25-Asn26, Leu67-Glu77, Ile99, and the Asp109-His110-Cys111 triad, and a distinct side-chain orientation of Asn53 was observed at the dimerization interface. While the β-barrel core remained rigid, these regions correspond to redox- and metal-responsive sites implicated in aggregation/fiber formation and putative drug binding.Conclusions: Temperature perturbs local dynamics in SOD1 structure without altering its native dimeric form. The ambient-temperature model reveals flexible, chemically accessible regions that act as druggable hotspots and coincide with ALS-linked mutation sites driving misfolding and aggregation. Considering temperature effects is crucial for structure-based drug design, ensuring candidate molecules engage physiologically relevant conformations. This structure lays the groundwork for future time-resolved crystallography of SOD1 folding and ligand interactions.