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. 2019 Oct 22;6(Pt 6):1099-1105.
doi: 10.1107/S2052252519011503. eCollection 2019 Nov 1.

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

Olivia Pfeil-Gardiner  1 , Deryck J Mills  1 , Janet Vonck  1 , Werner Kuehlbrandt  1
Affiliations

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

Olivia Pfeil-Gardiner et al. IUCrJ. .

Abstract

Radiation damage is the most fundamental limitation for achieving high resolution in electron cryo-microscopy (cryo-EM) of biological samples. The effects of radiation damage are reduced by liquid-helium cooling, although the use of liquid helium is more challenging than that of liquid nitrogen. To date, the benefits of liquid-nitrogen and liquid-helium cooling for single-particle cryo-EM have not been compared quantitatively. With recent technical and computational advances in cryo-EM image recording and processing, such a comparison now seems timely. This study aims to evaluate the relative merits of liquid-helium cooling in present-day single-particle analysis, taking advantage of direct electron detectors. Two data sets for recombinant mouse heavy-chain apoferritin cooled with liquid-nitrogen or liquid-helium to 85 or 17 K were collected, processed and compared. No improvement in terms of resolution or Coulomb potential map quality was found for liquid-helium cooling. Interestingly, beam-induced motion was found to be significantly higher with liquid-helium cooling, especially within the most valuable first few frames of an exposure, thus counteracting any potential benefit of better cryoprotection that liquid-helium cooling may offer for single-particle cryo-EM.

Keywords: apoferritin; beam-induced motion; beam-induced movement; cryo-EM; electron cryo-microscopy; helium; liquid-helium cooling; radiation damage.

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Figures

Figure 1
Figure 1
Exemplary micrographs with respective CTF estimations. (a) A micrograph acquired with liquid-nitrogen cooling, with an estimated defocus of 1.05 µm. The CTF goes out to 2.5 Å (white ring). (b) A micrograph acquired with liquid-helium cooling, with an estimated defocus of 1.99 µm. The CTF goes out to 2.6 Å (white ring).
Figure 2
Figure 2
Refined cryo-EM maps and slices of mouse heavy-chain apoferritin from data acquired with liquid-nitrogen cooling (a) at an estimated resolution of 2.74 Å and liquid-helium cooling (b) at an estimated resolution of 2.78 Å.
Figure 3
Figure 3
Fourier shell correlations (FSCs) of separately refined half maps from the data sets collected with lN2 cooling (FSC of 0.143 at 2.74 Å), lHe cooling (FSC of 0.143 at 2.78 Å) and between the two maps (FSC of 0.5 at 2.90 Å).
Figure 4
Figure 4
Histogram of defocus values of the particles used for reconstructions from data sets acquired with liquid-nitrogen or liquid-helium cooling.
Figure 5
Figure 5
Squared inverse resolution achieved from random subsets of increasing numbers of particles (logarithmic scale) with linear fits for data sets acquired with lN2 and lHe cooling. From the slope, an ‘overall B factor’ is determined.
Figure 6
Figure 6
Average per-frame motion of particles imaged with lN2 or lHe cooling as determined by the Bayesian polishing algorithm within RELION-3. With lHe cooling, the motion in the first few frames is higher by a factor of more than 2.
Figure 7
Figure 7
Selected carboxylate side chains showing signs of radiation damage. The reconstructed map from data acquired with lHe cooling (blue mesh) does not show better fits for the side chains of the fitted atomic model (PDB entry 3f32; Vedula et al., 2009 ▸) than the map from data acquired with lN2 cooling (red mesh).
Figure 8
Figure 8
Resolutions achieved by reconstructions using only single frames of the recorded movies, keeping the optimized angles and positions from refinements using all frames (top), and per-frame B factors as calculated with the Bayesian polishing function within RELION-3 (bottom) for data sets acquired with lN2 or lHe cooling.
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Grants and funding

This work was funded by Max-Planck-Gesellschaft grant .

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