Near-atomic resolution reconstructions from in situ revitrified cryo samples

We have recently introduced a microsecond time-resolved version of cryo-electron microscopy (cryo-EM) to enable the observation of the fast conformational motions of proteins. Our technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. We have previously described two alternative implementations of the technique, using either an optical microscope or performing revitrification experiments in situ. Here, we show that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within our spatial resolution. Interestingly, we observe that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation. SynopsisNear-atomic resolution reconstructions can be obtained from in situ melted and revitrified cryo samples. Revitrification results in a more homogeneous angular distribution.