Abstract text (incl. figure legends and references)
While cryo-electron microscopy (cryo-EM) is rapidly becoming the dominant method in structural biology, its time resolution is currently insufficient to directly observe proteins in action, leaving our understanding of these nanoscale machines fundamentally incomplete. Here, we demonstrate a novel approach to time-resolved cryo-EM that affords microsecond time resolution. Our method is illustrated in Fig. 1. It involves melting a cryo sample with a laser beam (a), which allows dynamics of the embedded particles to occur in liquid once a suitable stimulus is provided, for example by releasing a caged compound (b). While the dynamics occur, the heating laser is switched off at a well-defined point in time (c), causing the sample to rapidly recool, so that it vitrifies, and the particles are trapped in their transient configurations (d), in which they can subsequently be imaged (e). We demonstrate that our approach affords a time resolution of 5 µs or better. Moreover, near-atomic resolution reconstructions can be obtained from revitrified samples, showing that the revitrification process does not damage the protein structure. Finally, we present a microsecond time-resolved pH jump experiment, in which we observe the dynamics of the capsid of CCMV, an icosahedral plant virus. These results highlight the potential of our method to fundamentally advance our understanding of protein function through direct observation.
Figure 1. Experimental concept for microsecond time-resolved cryo-EM.