Structural transition of the ground-state structure to steady-state structures by sequential binding of ATP to V/A-ATPase

V/A-ATPase is a rotary ATPase that shares a common rotary catalytic mechanism with F _o F ₁ ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy (cryo-EM) during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. Here, we identified the cryo-EM structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of F _o F ₁ , where the overall structure of the F ₁ domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure. Our findings will enhance the future prospects for the initial activation processes of the enzymes with dynamical information, which contains unknown intermediate structures in their functional pathway.