High-resolution single-particle cryo-EM of samples vitrified in boiling nitrogen

Abstract

Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitrogen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitrogen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s−1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitrogen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitrogen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitrogen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

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

“…Since the cooling rate during revitrification is almost two orders of magnitude higher, one would therefore expect the beam-induced motion to become even more pronounced. We speculate that this is not the case since revitrification removes stresses that arise from large-scale deformations of the entire grid during plunge freezing (Engstrom et al, 2021;Thorne, 2020), which compensates for the additional local stress induced by the high cooling rate.…”

“…In particular, it is important to establish how it affects the beam-induced motion that occurs during imaging and that limits the obtainable spatial resolution. Such motion arises when mechanical stresses in the vitreous ice, which have built up during plunge freezing, are released under electron irradiation (Russo & Passmore, 2014; Naydenova et al ., 2020; Wright et al ., 2006; Brilot et al ., 2012; Zheng et al ., 2017; Engstrom et al ., 2021; Thorne, 2020). Figure 3a shows that the average cumulative drift of the apoferritin particles, which is displayed as a function of the electron dose, follows a typical behavior for both the conventional (grey) and revitrified cryo samples (purple, see Note S6 ).…”

“…It has been proposed that this stress results from the large temperature difference during plunge freezing between the holey gold film and the grid bars. Because of their large heat capacity, the bars cool more slowly and thus exert a compressive force on the vitreous ice film once they contract (Engstrom et al ., 2021; Thorne, 2020). Since melting and revitrification is highly localized and the grid bars remain at cryogenic temperatures throughout the entire process, this mechanism alone cannot easily explain the beam-induced motion of the revitrified samples (Voss et al ., 2021 a,b ).…”

“…Since melting and revitrification is highly localized and the grid bars remain at cryogenic temperatures throughout the entire process, this mechanism alone cannot easily explain the beam-induced motion of the revitrified samples (Voss et al ., 2021 a,b ). Our results are also seemingly at odds with the previous observation that drift increases with vitrification speed, which has been ascribed to the fact that faster cooling provides less time for stresses in the ice to dissipate during plunge freezing (Wu et al ., 2021; Engstrom et al ., 2021). Since the cooling rate during revitrification is almost two orders of magnitude higher, one would therefore expect the beam-induced motion to become even more pronounced.…”

Effects of cryo-EM cooling on structural ensembles

“…Above the liquid cryogen, a cold gas layer with a thickness of several mm was observed 74 , 75 . To what extent the cryo-EM samples are already cooled when they move through the gas layer during plunging is not clear.…”