Pushing Size and Resolution Limits of Single Particle Cryo-EM at 200 keV

Abstract

Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the 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 cryo-EM reconstructions of sub-100 kDa macromolecular complexes 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 and bound co-factors, features previously thought only to be resolvable using TEMs operating at 300 keV.