LAUSR

Na+,K+‐ATPase (NKA) is one of the most important members of the P‐type ion‐translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K+ state solved by cryo‐electron microscopy. It is a stable state with two occluded K+ after transferring three Na+ into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K+, linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K+ relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K+ into the cytoplasm, more than 100 times.