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 mutant does not appear to undergo the same steps as wild‐type P‐gp. We discuss conformational differences in the EQ mutant that may lead to a better understanding of the catalytic cycle of P‐gp and propose that additional structural studies with wild‐type P‐gp are required.