Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV

Abstract

Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

Figure 1. Cryo-EM reconstructions of the 20S proteasome at ~3.1 Å resolution

Local resolution estimates of the final T. acidophilum 20S proteasome reconstructions determined using either stage position (a) or image shift (c) navigation to exposure targets. An α-helix from the β-subunit (shown in stick representation) from each reconstruction with corresponding EM density (gray mesh, zoned 2 Å within atoms) exhibit clearly resolved side-chain density (b and d). (e) Gold-standard Fourier shell correlation curves indicate a final resolution of ~3.1 Å for the stage position dataset (black line) and ~3.3 Å for the image shift dataset (blue line). (f and g) The ~3 Å 20S EM density (gray mesh) is of sufficiently high resolution to observe ordered water molecules (shown as red spheres). Hydrogen bonds to the water molecules are shown as black dotted lines. (h) The EM density clearly shows that Tyr58 of the β-subunit adopts two alternative rotameric positions. The top 10 models refined against the density distribute into these rotameric positions, colored either orange (60%) or green (40%). (i) EMRinger analysis of the EM density corresponding to Tyr58 (blue line) confirms that the alternative conformations lie at ideal rotameric positions (thick gray bars). The refined χ¹ angles for Tyr58 are shown as vertical lines and colored according to panel C (conformation 1 is colored orange and conformation 2 is colored green).

Figure 2. Structure of aldolase at ~2.6 Å resolution

(a) The ~2.6 Å resolution aldolase EM density (D2 symmetric) segmented based on protomer organization. (b) The gold-standard Fourier shell correlation curve indicates a final resolution of ~2.6 Å at 0.143 FSC. (c) The local resolution estimate of the final aldolase reconstruction reveals that most of the molecule is resolved to better than 2.8 Å, with the core of the molecule resolved to ~2.5 Å. (d and e) Representative regions of the aldolase EM density (gray mesh, zoned 2 Å within atoms) indicate the map is of sufficient quality to unambiguously assign side-chain conformations as well as (f) the placement of putative ordered water molecules.