Microsecond Time-Resolved Cryo-Electron Microscopy

Proteins are the machinery of life and are therefore inherently dynamic systems. Yet, much of what we know about proteins still derives from static structures, leaving our understanding of their function fundamentally incomplete. Observing proteins in action requires not only atomic spatial resolution, but also a temporal resolution that matches the fast motions of proteins, a challenge that has largely remained beyond the reach of existing methods in structural biology. As part of a previous ERC Starting Grant, our group has established a novel approach to cryo-electron microscopy (cryo-EM) that promises to enable atomic-resolution observations of the dynamics of proteins with microsecond time resolution. This is notably fast enough to capture the large-amplitude domain motions of proteins that are typically associated with their function and thus promises to fundamentally change what we can know about proteins. However, significant efforts remain to take our technique beyond the proof-of-principle stage and to develop microsecond time-resolved cryo-EM into a mature technique that will be broadly adopted. This is the goal of the present proposal. To this end, we will overcome a number of technical challenges that currently limit the scope of our method. In particular, the observation window will be extended to longer timescales, and a variant will be implemented that affords nanosecond time resolution. In order for our work to have impact, it will also be crucial to ensure that the technique can be easily adopted by other labs. We will therefore develop a simpler implementation that does not rely on the highly specialized equipment only available in our lab. Finally, we will demonstrate that microsecond time-resolved cryo-EM can be used to elucidate the dynamics of a broad range of systems that have previously been inaccessible. To this end, we will study the dynamics of protein folding, the gating mechanism of an ion channel, and the dynamics of the SARS-CoV-2 spike protein.