P-type ATPases constitute a large family of primary ion transporters present in all kingdoms of life and generally responsible for maintaining ionic homeostasis. By transporting ions across cellular membranes, these molecules prime every type of cell for essential activities like muscle contraction, nutrient uptake and nervous impulses. Understanding their function and regulation at a fundamental level will give us insight into maintenance of normal cell function and how it goes wrong in various disease states, such as heart failure, dilated cardiomyopathy, diabetes and a variety of heritable syndromes. This proposal addresses of important representatives of this family of P-type ATPases, seeking first to define their molecular architecture, the conformational changes that characterize their reaction cycles, and the physical basis for their regulation by accessory proteins. We will use cryoelectron microscopy of two-dimensional crystals to determine structures and will interpret the resulting density maps by fitting homology models derived from existing x-ray structures of Ca-ATPase and of isolated cytoplasmic domains from various other family members. The P1b subfamily is characterized by N-terminal regions with specialized ion binding domains, and we will study several constructs of the representative CopA pump from Archaeoglobus fulgidis to identify the physical interactions of these domains with the rest of the molecule. The P1a subfamily is characterized by separate subunits for ATP hydrolysis and ion transport and appears to be directly descended from a primordial ancestor. Thus, we will study the P1a member, Kdp, to reveal the subunit architecture and to understand how energy coupling evolved in this family. Much is known about the P2a subfamily, given the recent success of x-ray crystallography on Ca-ATPase;nevertheless, we will study an isoform from scallop adductor muscle to reveal a native conformation of an important enzymatic intermediate in the absence of non-physiological inhibitors. We will address mechanisms of regulation of both Ca-ATPase and Na/K- ATPase by phospholamban and FxYD proteins, respectively. For phospholamban we will extend our current analysis to three-dimensions to reveal the fold of its cytoplasmic domain and interactions between monomers to form the pentamer. In addition, we will co-express the two proteins in insect cells to conduct structural studies under more physiologically relevant conditions. For FxYD proteins, we will develop conditions for 2D crystallization Na/K-ATPase and phospholemman after heterologous expression and purification from yeast.