Dynein, kinesin, and myosin, three classes of cytoskeletal motor proteins, power the majority of movements of eukaryotic cells. With regard to human health, many cardiac, kidney, auditory, and nervous system diseases have been linked to mutations in cytoskeletal motors. Small molecule drugs that manipulate the activities of myosin and kinesin motors (up-regulating cardiac myosin activity for heart failure or down-regulating mitotic kinesin activity for cancer) are now being tested in clinical trials. Of the thre types of cytoskeletal motor proteins, dynein is least well understood. While kinesin and dynein are both microtubule-based motor proteins, they have distinct structures and evolutionary histories; kinesin emerged from the G protein lineage, while the much larger dynein motor evolved from the AAA ATPases. The goal for this grant is to understand the structural basis of motility by cytoplasmic dynein, the motor that drives the vast majority of minus-end-directed microtubule motility of intracellular cargoes such as membranes, mRNAs, chromosomes and viruses. Our past grant focused on X-ray crystallography of dynein. While we will continue to utilize this approach, we are shifting more towards electron microscopy because of recent advances in cryo EM that can produce structures with atomic resolution. We are collaborating with an investigator at UCSF who is pioneering such approaches. With these tools, we propose to solve structures for dynein in its "pre-powerstroke" states, complementing earlier X-ray structures of the "post-powerstroke" states. Such work will complete our view of dynein's chemomechanical cycle, allowing us to understand how transitions in the ATPase cycle trigger allosteric changes across the large dynein motor domain which produces motility. To complement these structural "snap shots", we will make dynamic measurements of the structural changes in active, cycling dynein motors using single molecule techniques. We also will dissect the roles of dynein's three active ATPase sites using pre- steady state nucleotide binding measurements. Kinesin and myosin only have a single ATPase site, so we hope to resolve the mystery of how dynein utilizes its two additional ATPase sites. The above work will be performed with the yeast dynein motor domain, which is constitutively active and a good model system for understanding dynein motility. However, we recently discovered that mammalian cytoplasmic dynein is more complicated and requires a cargo adapter protein and dynactin (a multi-subunit protein complex) to become fully active. To understand the structural basis of this regulatory mechanism, we will obtain a cryo EM structure of the large dynein-dynactin-adapter complex in order to understand how these components interact and how these interactions lead to dynein activation. Thus, by the end of this grant period, we hope to derive a detailed model for the structural changes that drive dynein motility and illuminate a still poorly understood mechanism for regulating dynein in mammalian cells.