LAUSR

Significance The E2P state of Na+,K+-ATPase, in which the ATPase is phosphorylated and of low affinity to Na+ with the extracellular gate opened, shows different biochemical properties depending on whether the phosphate is transferred from ATP in the forward reaction or from inorganic phosphate (Pi) in the backward reaction. We present here cryoelectron microscopy structures of Na+,K+-ATPase in the two E2P states, explaining their different biochemical properties established a half century ago. The new electron microscopy maps show previously unseen structural features, including unexpected binding modes of cardiotonic steroids, specific and medically important inhibitors of the ATPase, and stabilization by ATP of the E2P state.