Muscle contraction is a central feature of animal life. It results from interactions between myosin heads on the thick filaments and actin subunits on the thin filaments. While great progress has been made in understanding the molecular basis of contraction and its regulation, key questions remain. These include the structural basis of regulation of myosin and actin filaments, and the molecular basis of actin-myosin interaction. To elucidate these mechanisms it is crucial to determine the structures of the filaments in relaxed and activated states and to capture dynamic molecular changes. We do this using state-of-the-art cryo-EM and new millisecond time-resolved techniques, combined with 3D image reconstruction. Using these approaches: (1) We will determine the structural basis of calcium- and phosphorylation-based regulation of striated muscle myosin filaments by defining the molecular interactions occurring in isolated myosin molecules and filaments. (2) We will define the structural basis of thin filament regulation of striated muscle by analyzing troponin, tropomyosin and actin interactions at high and low Ca 2+, the dynamics of tropomyosin movement, structural alterations in myopathies, and the organization of the thin filament template protein, nebulin. (3) We will define the binding sites of myosin heads on actin at early stages of the actomyosin crossbridge cycle by analyzing thin filaments decorated with weakly bound myosin heads. In each study, reconstructions will be fitted to atomic resolution maps of F-actin, troponin or the myosin head, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insight into general mechanisms of thick filament regulation. Continued advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin, and the importance of tropomyosin dynamics in thin filament regulation and disease. Studies of the actomyosin complex will directly test models for the interaction of myosin heads with actin during the fundamental crossbridge cycle. Preliminary data demonstrate that all of our proposals are feasible. New insights arising from these proposals will provide a deeper understanding of the structural basis of muscle contraction and its regulation, and of actomyosin-based cell motility. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects in skeletal and cardiac myopathies.