Multiple sub-state structures of SERCA2b reveal conformational overlap at transition steps during the catalytic cycle

Sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps Ca²⁺ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca²⁺-bound phosphorylated (E1P·2Ca²⁺) and Ca²⁺-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca²⁺ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca²⁺ and that the Ca²⁺ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.