Changes within the central stalk of E. coli F1Fo ATP synthase observed after addition of ATP

F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the F-o motor that is transferred to the F-1 motor to catalyze ATP production, with flexible F-1/F-o coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory epsilon subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory epsilon subunit induce torsional movement in the central stalk, thereby enabling its rotation within the F-omicron motor. This highlights the importance of the central rotor for flexible coupling of the F-1 and F-o motors and provides further insight into the regulatory mechanism mediated by subunit epsilon.