The basal cycle (middle panels, steps 1 and 2) entails conformational sampling by ATP-bound Pgp to enable NBD dimerization followed by population of a symmetric HES. The substrate-coupled cycle (steps 1, 3a, 4a, and 2) is initiated by substrate binding. As previously postulated (8, 16, 18), a transient conformation with one tightly bound ATP molecule is likely populated. Hydrolysis of one ATP stabilizes the asymmetric HES (step 4a), which consists of OO/OF conformations. Hydrolysis of the second ATP leads to the symmetric HES as in the basal cycle (step 2). Pgp inhibition (steps 1, 3b, and 4b) is initiated by stabilization of an IF conformation in which the extracellular sides of TMD is apo-like, whereas the intracellular sides of TMDs and NBDs are closer than the apo state. ATP hydrolysis proceeds through a heterogeneous HES. For each intermediate, we show cytoplasmic views of the NBDs highlighting the conserved A-loop tyrosines as purple spheres along with associated DEER distributions in nanodiscs.