High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our [~]2.9 [A] structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our [~]2.8 [A] and [~]3.2 [A] resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.