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

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.

Proteorhodopsin: characterisation of 2D crystals by electron microscopy and solid state NMR.

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in gamma-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.