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P-glycoprotein (P-gp, ABCB1) is an ABC transporter associated with the development of multidrug resistance to chemotherapy. During its catalytic cycle, P-gp undergoes significant conformational changes. Recently, atomic structures of some of these conformations have been resolved using cryo-electron microscopy. The ATP hydrolysis-defective mutant of the catalytic glutamate residue of the Walker B motif (E556Q/E1201Q) has been used to determine the structure of the ATP-bound inward-closed conformation of P-gp. Here, we show that this mutan