Advances in Microsecond Time-Resolved Cryo-Electron Microscopy

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution [1,2], about three orders of magnitude higher than that of traditional approaches. Importantly, this is fast enough to access a wide range of protein dynamics that have previously eluded observationamong them the large-scale domain motions that are typically associated with the activity of a protein and that frequently occur on microsecond timescales. Our method therefore promises to fundamentally advance our understanding of protein function through the direct observation of the dynamics involved. Our technique involves rapidly melting a cryo sample with a laser beam. Once the sample is liquid, conformational dynamics of the embedded particles are initiated by providing a suitable stimulus. As these dynamics unfold, the laser beam is then switched off at a well-defined point in time, so that the sample rapidly cools and revitrifies, trapping the particles in their transient configurations, in which they can subsequently be imaged. We have previously demonstrated that the revitrification process, which determines the time resolution of the experiment, occurs in just a few microseconds [1]. Moreover, it preserves the structure of the proteins [3], so that near-atomic resolution reconstructions can be obtained from revitrified cryo samples [4,5]. This suggests that our method does not fundamentally limit the obtainable spatial resolution. Here, we report on several recent advances of our technique that we have implemented. In order to make microsecond timeresolved cryo-EM accessible to a wide range of researchers, we have established a simplified experimental approach. While we have previously performed experiments in situ with a modified transmission electron microscope, few other labs have such specialized equipment at their disposal. Here, we show that melting and revitrification experiments can instead be carried out in an optical microscope, using a correlative light-electron microscopy approach. Optical microscopes are already available in many cryo-EM labs. Moreover, they are more cost-effective as well as easier to modify. We therefore hope that this will encourage others to adopt our technique. We observe that melting and revitrification reshuffles the particles in the cryo sample, scrambling their orientations in the process [4]. This suggests that it may be possible to employ revitrification as a means to alleviate issues with preferred particle orientation, which in some samples severely limits the obtainable resolution of single-particle reconstructions. We speculate that the reshuffling of the particles may be related to partial crystallization of the sample during the laser melting process. Finally, we report on a microsecond time-resolved study of the capsid dynamics of cowpea chlorotic mottle virus (CCMV). This icosahedral plant virus undergoes a large-scale expansion as it enters the host cell,