A comparative study of single-particle cryo-EM with liquid-nitrogen and liquid-helium cooling

Abstract

Radiation damage is the most fundamental limitation for achieving high resolution in cryo-EM, and is expected to be reduced at liquid-helium temperature. Surprisingly, cryo-EM reconstructions of apoferritin samples cooled with liquid helium showed no improvement in resolution over liquid-nitro­gen-cooled samples, but showed substantially more beam-induced particle motion.

Devitrification reduces beam-induced movement in cryo-EM

“…To take full advantage of the improved stability of the cubic ice lattice, it would be desirable to devitrify specimens on all-gold grids (Russo & Passmore, 2014), which are considerably more stable than Quantifoil copper grids, and to image them in a liquid helium-cooled microscope at a temperature between 4 and 17 K. Cooling with liquid helium is known to protect 2D crystals of bacteriorhodopsin against radiation damage by a factor of $2 in electron dose compared with liquid nitrogen (Stark et al, 1996;Vinothkumar & Henderson, 2016). For single particles, this potential benefit of helium cooling is neutralized by a large, unexpected increase in beam-induced specimen movement (Pfeil-Gardiner et al, 2019), but joint use of specimen devitrification and liquid-helium cooling might overcome this problem. Figure 7 20 000 particles were randomly chosen from the final 3D refinement of the full data sets and reextracted from individual frames for 3D reconstruction.…”

Current limitations to high-resolution structure determination by single-particle cryoEM

“…Beam-induced movement of cryoEM specimens occurs in two phases (Fig. 4) (Russo and Passmore, 2014 b ; Pfeil-Gardiner et al ., 2019). The initial phase is characterized by a burst-like motion, as the first electrons hit the specimen, releasing the stress trapped in the vitreous layer, and the doming becomes more pronounced.…”

“…Surprisingly, the movement in both phases is worse by a factor of two or more for samples cooled with liquid helium to around 4–17 K, rather than with liquid nitrogen to 84 K (Fig. 4 a ) (Pfeil-Gardiner et al ., 2019). For 2D crystals, cooling with liquid helium is known to reduce radiation damage by roughly a factor of two (Kühlbrandt et al ., 1994; Stark et al ., 1996).…”

“…For 2D crystals, cooling with liquid helium is known to reduce radiation damage by roughly a factor of two (Kühlbrandt et al ., 1994; Stark et al ., 1996). It would be excellent if we could take advantage of this extra cryoprotection factor for single particles as well, but at present, the massive beam-induced movement observed with non-crystalline, unsupported particles (Pfeil-Gardiner et al ., 2019) counteracts and obliterates the potential beneficial effect of helium cooling. Once this puzzling problem is resolved, liquid helium cooling should improve the signal-to-noise ratio of low-dose single-particle images just as much as for 2D crystals.…”

“…4.Beam-induced movement of protein complexes. ( a ) Histogram of averaged per-frame particle movements in apoferritin specimens cooled with liquid nitrogen to 85 K (red) or liquid helium to 17 K (Pfeil-Gardiner et al ., 2019). ( b ) Averaged beam-induced per-frame particle movements of alcohol oxidase particles in vitrified (blue) and devitrified (orange) layers of solid water.…”