Protocol for purification, single-particle cryo-EM structure determination, and functional assays of the fungal plasma membrane H+-ATPase

The fungal plasma membrane H⁺-ATPase (Pma1) maintains intracellular pH and membrane potential, but its study has been limited by challenges in sample preparation and functional assays. Here, we present a protocol for the endogenous purification of Pma1 in Saccharomyces cerevisiae, assessment of ATPase activity, cryo-electron microscopy (cryo-EM) structure determination, generation of a conditional pma1 knockout strain, and the performance of growth assays on mutants. For complete details on the use and execution of this protocol, please refer to You et al.¹.