Structure of β-galactosidase at 3.2-Å Resolution Obtained by Cryo-Electron Microscopy

The vast majority of high-resolution structures obtained using cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or two-dimensional crystals. We show here that emerging methods in single-particle cryo-EM now allow structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry, by determining the structure of the 465-kDa enzyme β-galactosidase. In addition, by quantitative comparison of density maps obtained at different electron dosages, we demonstrate preferential sensitivity of residues such as Asp and Glu to damage upon irradiation with electrons.

Proc Natl Acad Sci, 111(32):11709–14, 2014.


We report the solution structure of Escherichia coli β-galactosidase (~465 kDa), solved at ~3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ~0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e−/Å2. In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.