Residue Specific Radiation Damage of Protein Structures using High-Resolution Cryo-Electron Microscopy

Abstract

High-resolution structures of proteins and protein complexes are currently determined using either X-ray crystallography, NMR spectroscopy, or now also cryo-electron microscopy (cryo-EM). The highest resolutions achieved by cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or 2D crystals. However, we show 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. We solved the structure of the ∼465-kDa Escherichia coli β-galactosidase at ∼3.2-Å resolution using single-particle cryo-EM. At this resolution, the majority of the side-chains, the N-termini, and the geometry of the active sites, including a catalytic Mg2+-ion, can be clearly discerned in the density map. Inspection of the map reveals that while 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. The negatively charged glutamate and aspartate show on average 30% less density than the similarly sized neutral glutamine and asparagine. This observation is independent of the exposure of these residues to solvent. Analysis of other high-resolution cryo-EM structures reveals similar weaker densities for these types of residues. Radiation damage in X-ray crystallography has been linked to decarboxylation of glutamate and aspartate residues, breakage of disulfide bonds, loss of hydroxyl-groups from tyrosine and methylthio-group of methionine. We now show that negatively charged residues exhibit more pronounced effects of radiation damage in structures solved by cryo-EM. We determined that the degree of damage is dose dependent by comparison of density maps obtained using electron doses ranging from 10-30 electrons/Å2. In summary, we establish the feasibility of determining structures at near-atomic resolution and provide a measure of the effects of radiation damage in high-resolution cryo-electron microscopy.