Technical Highlight - May 2015
Short description: The 2.8-Å resolution cryo-EM reconstruction of a thermophilic 20S proteasome identifies rotamers and water molecules.
Since its early days, structural biology has been identified primarily with X-ray crystallography. Indeed, about 96,000 of 110,000 structures deposited in the PDB to date were determined by crystallography. One reason for this success is that crystallography allows for atomic resolutions with virtually no limits in protein size. Cryo-electron microscopy (cryo-EM) has two major advantages over crystallography: determination of protein structures in solution-like conditions and structural characterization of heterogeneous complexes. However, cryo-EM has been hampered by the significantly lower resolution it can reach compared to crystallography.
Recent technical improvements have allowed a giant leap in resolution for cryo-EM. New direct-electron detectors are supplanting charge-coupled device cameras, with higher signal-to-noise ratios and faster acquisition rates that permit recording of multiple images (movies) during the same exposure times. Newly developed algorithms enable correction of sample movements induced by the electron beam, with further improvement of signal-to-noise ratio. These advances have resulted in a surge of cryo-EM reconstructions below 4-Å resolution. Carragher and colleagues have now reported the cryo-EM reconstruction of the Thermoplasma acidophilum 20S proteasome (T20S) at 2.8-Å resolution.
The authors collected data at full 53 e−/Å2 exposure, then carefully selected micrographs with Thon rings visible up to 4-Å resolution or better in their power spectra. They sorted about 87,000 particles (selected from ∼200 movies, each containing 38 frames), which were used for a first round of reconstruction yielding 2.98-Å resolution. Subsequent particle polishing to account for motions due to beam exposure improved the resolution to 2.83 Å. Elimination of particle images with the highest angular uncertainty eventually pushed the reconstruction to 2.81-Å resolution. The authors then built the atomic model by rigid-body fitting the crystal structure of T20S (PDB 1YAR) into the cryo-EM map, performed iterative refinement with Rosetta and improved the quality of the model by manual adjustment using Coot.
The quality of the T20S proteasome cryo-EM map is akin to X-ray crystallography structures at comparable resolution, and allows unambiguous assignment of side chains (e.g. distinguishing between Phe and Tyr), establishment of different rotameric conformations and identification of ordered water molecules. This level of details puts cryo-EM on par with X-ray crystallography for de novo determination of protein structures.
M.G. Campbell, D. Veesler et al. 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy.
eLife. 4 (2015). doi:10.7554/eLife.06380