Credit: CC BY 4.0

Substantial technical advancements have recently allowed single-particle cryo-EM to achieve resolutions comparable to those of X-ray crystallography in protein-structure determination. New direct electron detectors with faster acquisition rates and higher signal-to-noise ratios are supplanting charge-coupled-device cameras, and new algorithms enable correction of sample movements induced by the electron beam, to further boost the signal-to-noise ratio. These developments have led to a surge of cryo-EM–based protein reconstructions below 4-Å resolution. Carragher and colleagues have now reported the cryo-EM reconstruction of a protein complex below 3-Å resolution, for the Thermoplasma acidophilum 20S proteasome (T20S). By multiple rounds of particle selection to account for motions due to beam exposure and to eliminate particle images with high angular uncertainty, they sorted ∼50,000 particles for T20S reconstruction at 2.81-Å resolution. Carragher and colleagues built the atomic model by rigid-body fitting of the crystal structure of T20S into the cryo-EM map, performing iterative refinement with Rosetta and improving the model by manual adjustments with Coot. The quality of the T20S cryo-EM map is akin to that achieved by X-ray crystallography at comparably high resolution, and it allows unambiguous assignment of side chains, establishment of different rotameric conformations and identification of ordered water molecules. This level of detail opens the possibility for cryo-EM to compete with X-ray crystallography for de novo determination of protein structures. (eLife doi:10.7554/eLife.06380, 11 March 2015)