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

Abstract

Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, 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 high-resolution structures of specimens amassing less than 100\u2009kDa using a transmission electron microscope operating at 200\u2009keV coupled with a direct electron\xa0detector. Our ~2.7\u2009A structure of alcohol dehydrogenase (82\u2009kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8\u2009A and ~3.2\u2009A structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64\u2009kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein. Despite many recent advances in cryo-EM, imaging smaller macromolecules (below 100\u2009kDa) has remained a challenge. Here\xa0the authors show that biological specimens amassing <100\u2009kDa can be resolved to better than 3\u2009A resolution using conventional defocus-based single-particle analysis methods.