Recovery of a minor cryo-EM particle population reveals conformational equilibria linking cofactor loading, turnover, and reactivation in methionine synthase.

Cobalamin-dependent methionine synthase (MetH) is a highly dynamic enzyme that requires large-scale domain rearrangements to facilitate turnover and reactivation. Despite significant biochemical and structural efforts, a full picture of the conformational changes underlying the transition between MetH turnover and reactivation has remained elusive. Here, we leverage state-of-the-art cryo-electron microscopy (cryo-EM) methods to reanalyze an old dataset and gain new insights into these changes. By utilizing a neural-network particle picker, performing careful classification of particles, and incorporating high-resolution information to determine initial particle orientations, we uncovered and reconstructed a minor population of reactivation-state MetH (∼4.5-5 Å resolution) from a dataset that is dominated by resting-state MetH. This structure represents the first fully intact structure of the enzyme with a physiological cobalamin cofactor bound. These findings highlight the potential for improved cryo-EM methodology to uncover previously hidden details about enzyme function and deepen our understanding of structural ensembles.