Pre-catalytic activation pathway of the Smc5/6 complex

The Smc5/6 complex (Smc5/6) preserves genome stability through ATP-dependent DNA organization, but the structural details that prime the complex for productive DNA engagement remain unknown. Here, we combine cryo-electron microscopy and conformational heterogeneity analysis to define the pre-catalytic activation pathway of Smc5/6. We show that in the absence of ATP and DNA, Smc5/6 exists as a heterogeneous conformational ensemble composed predominantly of two conformations termed Bridge and Cross. In the presence of DNA alone, Smc5/6 adopts a Juxtaposed conformation that brings the ATPase heads into proximity, aligning them for nucleotide engagement. With both ATP and DNA associated, Smc5/6 establishes an Engaged architecture, within which DNA stabilizes its head-associated binding cavity through local remodeling of the complex. Conformational heterogeneity analyses suggest a continuous structural transition between the Cross and Juxtaposed states, while anisotropic network modeling identifies the Bridge–Cross–Juxtaposed–Engaged sequence as the mechanically favored pathway connecting the four experimentally observed conformations. Together, these findings support a pre-catalytic conformational pathway for Smc5/6 activation and provide a structural framework for understanding how conformational heterogeneity gives rise to an activation-competent motor state.