Completing the myosin mechanochemical cycle with time-resolved cryoEM

Structural biology has undergone a revolution in its power with fundamental techniques such as cryoEM and X-ray crystallography revealing higher resolution detail on increasingly more complex targets. In parallel, programs such as alpha-fold now produce ever more accurate computational predictions of protein structure. Whilst these all provide important details on protein structure that underpins our fundamental understanding of biology and drive the development of new therapeutics they are typically a “snapshot” and lack “temporal” resolution that informs on the mechanistic steps of a protein or protein complex. Time-resolved approaches bring the temporal resolution to structural biology by typically triggering a reaction and then stopping the reaction along its pathway before completion to understand the mechanism of action and associated conformational change. One such method, time-resolved cryoEM, permits the trapping of large conformational changes in proteins and is a rapidly growing field. This proposal will use and develop time-resolved cryoEM to trap the molecular motor myosin-5, and other members of the myosin family, in a series of different states to provide a step change in our understanding of how the myosin motor functions. This is key to understanding how myosin motors perform a range of functions in eukaryotes, unlocking why certain mutations can cause disease, such as early onset cardiac disease, and how small molecules modulate function. This will be done through three specific objectives: Objective 1 will build on our structure of the primed acto-myosin-5 structure (where the motor is primed ready to move across its actin track and was trapped in ~10 milliseconds) to improve the resolution and allow us to look at the details of how the ATP fuel for the motor is utilised. We collected our original data in 2020 and we will take advantage of improvements in technology and our increased experience of undertaking time-resolved experiments to improve resolution. Objective 2 will solve the structure of a previously unknown state where the myosin-5 motor is bound to its track and ATP. After binding ATP myosin-5 dissociates from its track in less than one millisecond and therefore a new system for time-resolved EM that can go ~10 times faster than previous approaches will be developed. Objective 3 will look at different molecular motors of the myosin super family (myosin-2 and -6) to understand how conserved the mechanism of action of myosin-5 is over the wider family so we can start to understand the “rules of life” for this important motor superfamily and not just one specific member. These objectives are based on strong pilot data that show we can achieve each step, our ability to produce the protein required, and our experience with time-resolved methodologies. The unique nature of which make us uniquely placed to conduct this important research. Moreover, the expanding nature of the time-resolved field means that expertise are urgently sought within the UK workplace. We will mentor and train the postdoctoral researcher to become a leader in this field and run a workshop to train the wider community, alongside presenting our technology at regular EM training events internal and external to Leeds. Beyond the academic arena we will liase with industry on both methodology expansion and possible equipment licensing and our public engagement activities will ensure the work reaches a wide audience.