The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E-1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1P.ADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E-1 to the E1P.ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E-1 and E1P.ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E-1 and E1P.ADP states present in the 2D crystals, showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase. (c) 2007 Elsevier Inc. All rights reserved.