Microsecond Melting and Revitrification of Cryo Samples – Protein Structure and Beam-Induced Motion

Abstract

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time-resolution of just a few microseconds, this approach is notably fast enough to study domain motions that are typically associated with the activity of proteins, but which have previously remained inaccessible. Here, we add crucial details to the characterization of our method. We show that single-particle reconstructions of apoferritin and cowpea chlorotic mottle virus (CCMV) from revitrified samples are indistinguishable from those in conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by our instrument. We also characterize how rapid revitrification affects the properties of the ice, showing that revitrified samples exhibit comparable amounts of beam-induced motion. Our results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and address beam-induced specimen movement.SynopsisMicrosecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional cryo samples.

Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach

“…By adjusting the laser power to keep the diameter of the revitrified area constant, one can thus ensure that in each experiment, the sample undergoes the same temperature evolution (Voss et al, 2021a). We have also demonstrated that the melting and revitrification process leaves the proteins intact (Harder et al, 2022). Near-atomic resolution reconstructions can be obtained from revitrified cryo samples, suggesting that the revitrification process does not fundamentally limit the obtainable spatial resolution (manuscript in preparation; see also Lorenz, U. J., 2022).…”

“…This suggests that the revitrification process does not fundamentally limit the obtainable spatial resolution. Revitrification with an optical microscope is in several respects complementary to the in situ approach we have previously described (Voss et al, 2021b;2021a;Harder et al, 2022), with each approach offering distinct advantages. It is usually more straightforward to determine the diameter of the revitrified area in an in situ experiment, since the surrounding crystalline region offers a strong contrast in an electron microscope, whereas it may be more difficult to detect in optical difference images if the crystallites are too small.…”

“…Melting and revitrification can be used to turn some crystalline areas vitreous or reduce the ice thickness through evaporation. It may also be possible to reduce beam induced specimen motion by releasing stress in the vitreous ice film through irradiation with a sequence of laser pulses (Harder et al, 2022).…”

“…We have recently introduced a novel approach to timeresolved cryo-EM that affords microsecond time resolution (Voss et al, 2021b;2021a;Harder et al, 2022). This is notably fast enough to enable the observation many domain motions.…”