Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F 1 -F o coupling

F 1 F o -ATP synthases play a central role in cellular metabolism, making the energy of the proton-motive force across a membrane available for a large number of energy-consuming processes. We determined the single-particle cryo-EM structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at 2.7- 2.8 Å resolution. Separation of 13 well-defined rotary substates by 3D classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F 1 head rotates along with the central stalk and c-ring rotor for the first ~30° of each 120° primary rotary step. The joint movement facilitates flexible coupling of the stoichiometrically mismatched F 1 and F o subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit, a well-established target of physiologically important inhibitors. Our maps provide atomic detail of the c -ring/ a -subunit interface in the membrane, where protonation and deprotonation of c -ring c Glu111 drives rotary catalysis. An essential histidine residue in the lumenal proton access channel binds a strong non-peptide density assigned to a metal ion that may facilitate c -ring protonation, as its coordination geometry changes with c -ring rotation. We resolve ordered water molecules in the proton access and release channels and at the gating a Arg239 that is critical in all rotary ATPases. We identify the previously unknown ASA10 subunit and present complete de novo atomic models of subunits ASA1-10 , which make up the two interlinked peripheral stalks that stabilize the Polytomella ATP synthase dimer.