Cryo-EM Structural Mechanism of a 2-Oxoglutarate-Dependent Dioxygenase at 2.4 Angstrom Resolution: Substrate Loop Ordering, Iron Coordination Geometry, and Catalytic Cycle Intermediates

This study investigates cryo-EM structure determination at 2.4 A resolution of a 2-oxoglutarate-dependent dioxygenase in apo, substrate-bound, 2OG-bound, and transition state analog-bound states revealing active site loop ordering and Fe(II) coordination geometry changes along the catalytic cycle within the context of structural biochemistry and enzymology, an area of growing scientific importance given its implications for m6A RNA modification drug target characterization, 2-oxoglutarate dioxygenase family inhibitor design, and cryo-EM methodology for enzyme mechanism studies. Using cryo-EM vitrification (Vitrobot Mark IV), data collection on Titan Krios G4 (300 kV, Falcon 4 detector), RELION-4 processing with CTF refinement and Bayesian polishing, PHENIX real-space refinement against density, and MD simulation for loop dynamics, we examine substrate RNA binding triggering active site loop closure that displaces axial water from Fe(II) coordination sphere, enabling 2-oxoglutarate bidentate coordination to create reactive ferryl (Fe(IV)=O) intermediate for N-methyl group oxidation in m6A demethylation in 4 cryo-EM datasets: apo (84,200 particles), RNA-bound (62,400 particles), 2OG-bound (72,800 particles), NOG-bound (58,400 particles); final maps at 2.4, 2.8, 2.6, 2.4 A resolution respectively drawn from Pacific Biochemistry Institute Titan Krios G4 at 300 kV with Falcon 4 direct electron detector, automated data collection by EPU software, and RELION 4.0 high-performance computing cluster. Results indicate that RNA binding induces 8.4 A loop closure (RMSD 2.84 A vs. apo), reducing active site volume 42.4%; Fe(II) coordination shifts from octahedral (6-coordinate, water-occupied) to square pyramidal (5-coordinate) upon RNA+2OG binding, primed for O2 activation; NOG captures near-attack conformation with Fe-N distance 2.24 A (p < 0.001), with 8.4 A loop closure; 42.4% active site volume reduction; Fe coordination shift to 5-coordinate as the primary quantitative benchmark. Concordance between primary and confirmatory measurement approaches exceeded 93%, validating the analytical framework. These findings contribute empirically to structural biochemistry and enzymology and carry actionable implications for the design of programs and policies targeting m6A RNA modification drug target characterization, 2-oxoglutarate dioxygenase family inhibitor design, and cryo-EM methodology for enzyme mechanism studies.