Microsecond melting and revitrification of cryo samples

Abstract

The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.

Microsecond melting and revitrification of cryo samples: protein structure and beam-induced motion

“…1(e), a circular area around the center of the laser focus has melted and revitrified (dashed semicircle). In contrast, the adjacent regions, in which the temperature has remained below the melting point of water, have crystallized (Voss et al, 2021a). We then collect micrographs from holes within the revitrified area, such as that in Fig.…”

“…We note that in order to ensure a comparable temperature evolution in all melting and revitrification experiments, the laser power (typically about 165 mW) was always adjusted to obtain a revitrified region of similar size to that in Fig. 1(e) (Voss et al, 2021a). We note that while aligning the laser the sample is exposed to a dose of about 10 À3 electrons A ˚À2 before it is melted and revitrified.…”

“…The melting step resembles the revitrification process in that it occurs on the same timescale of a few microseconds, just with the temperature evolution reversed (Voss et al, 2021b). One notable difference, however, is that cubic ice forms during the early stages of laser heating, which only melts once the temperature exceeds 273 K (Voss et al, 2021a). While the formation of cubic ice crystals could conceivably damage the proteins, this does not appear to be the case.…”

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

“…We then collect micrographs from holes within the revitrified area, such as that of Figure 1f, which reveals intact apoferritin particles. We note that in order to ensure a comparable temperature evolution in all melting and revitrification experiments, the laser power (typically about 165 mW) was always adjusted to obtain a revitrified region of similar size to that in Figure 1e (Voss et al, 2021a).…”

“…As indicated in Figure 1e, a circular area around the center of the laser focus has melted and revitrified (dashed semicircle). In contrast, the adjacent regions, in which the temperature has remained below the melting point of water, have crystallized (Voss et al, 2021a). We then collect micrographs from holes within the revitrified area, such as that of Figure 1f, which reveals intact apoferritin particles.…”