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. 2021 Sep 7;8(Pt 6):867-877.
doi: 10.1107/S2052252521008095. eCollection 2021 Nov 1.

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

Tyler Engstrom  1 , Jonathan A Clinger  2 , Katherine A Spoth  3 , Oliver B Clarke  4   5 , David S Closs  1 , Richard Jayne  1 , Benjamin A Apker  1 , Robert E Thorne  1   2
Affiliations

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

Tyler Engstrom et al. IUCrJ. .

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 nitro-gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro-gen 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 nitro-gen 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 nitro-gen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitro-gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

Keywords: cryocooling; cryoelectron microscopy; vitrification.

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Figures

Figure 1
Figure 1
(a) and (b) Real space detector images and (c) and (d) corresponding diffraction mode detector images of a biomolecule-free 0.5% NaCl solution on a 400 mesh Quantifoil holey carbon grid with 2 µm holes plunge-cooled in boiling LN2. Both thick and thin ice were fully vitrified with no ice diffraction evident. The dashed lines in (c) and (d) indicate the expected positions of pure cubic ice diffraction (orange lines) at 1/d = 2.73, 4.45 and 5.22 nm−1 and of stacking disordered ice diffraction which, in addition to the peaks of cubic ice, typically has additional strong peaks at the hexagonal ice positions 2.57 and 2.91 nm−1 (yellow lines).
Figure 2
Figure 2
Sample real space detector images and corresponding image CTFs and FFTs of 5 mg ml−1 apoferritin solutions that were dispensed and blotted on (a) and (b) a Quantifoil UltraAuFoil grid (sample 1) and (c) and (d) a prototype grid (sample 2), both having gold foils with 1.2 µm holes, and then plunge-cooled in boiling LN2. Sample film thicknesses and areal particle densities were typically larger for sample 1 than sample 2. The majority of hole images and FFTs for sample 1 showed evidence of small amounts of crystalline ice, whereas nearly all hole images for sample 2 were fully vitrified.
Figure 3
Figure 3
Single-particle reconstruction and refined model based on apoferritin data obtained from sample 1, which was deposited on a Quantifoil grid and plunge-cooled in boiling LN2. (a) Apoferritin model placed into surface map representation. Ribbons of apoferritin monomers colored by chain designation. (b) Single monomer of apoferritin showing map-monomer fit. (c) Apoferritin helix comprised of residues 132–154 demonstrating the sidechain fit. Maps in (a)–(c) are contoured at 1σ. The Fourier shell correlation (FSC) plot is shown in Fig. S7(a).
Figure 4
Figure 4
Beam-induced motion, sample thickness and ice for (a) sample 3 and (b) sample 4. Shown are (i) an image of a foil hole at a fluence of 1.00 e− Å−2; (ii) particle positions measured in the first and fifth frames corresponding to fluences of 0.55 and 5.5 e− Å−2, respectively; (iii) sample film thickness map determined by comparing transmitted intensities with and without an energy slit; and (iv) diffraction mode image (left) and FFT of real space image (right).
Figure 5
Figure 5
Drift-corrected RMS particle displacement versus fluence, all measured using the same cryo-TEM. Grid type A is Quantifoil UltraAuFoil 1.2/1.3, Au foil with 1.2 µm holes on Au grids; type B is prototype Au foil with 1.2 µm holes on a Cu grid; type C is prototype Au foil with 1.2 µm holes on an Au grid. Samples 1–6 were cooled in boiling LN2, and sample 7 was cooled in ethane using a Vitrobot Mark IV. Samples 1 and 3 were partially vitrified; most holes gave good particle images but showed local ice, confirmed by appreciable intensity at ice ring positions in image FFTs. Samples 2 and 3–7 were largely vitrified with only a small minority of frames showing evidence of ice.
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References

    1. Bald, W. B. (1984). J. Microsc. 134, 261–270.
    1. Bald, W. B. (1985). J. Microsc. 140, 17–40.
    1. Bald, W. B. (1986). J. Microsc. 143, 89–102. - PubMed
    1. Berejnov, V., Husseini, N. S., Alsaied, O. A. & Thorne, R. E. (2006). J. Appl. Cryst. 39, 244–251. - PMC - PubMed
    1. Brüggeller, P. & Mayer, E. (1980). Nature, 288, 569–571.