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...