Journal Article

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

Wu, Mengyu, Herzik, Mark A., Lander, Gabriel C.

2019

نظرة عامة

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 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å 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 sub-nanometer cryo-EM structure of a sub-50 kDa protein. Despite many recent advances in cryo-EM, imaging smaller macromolecules (below 100 kDa) has remained a challenge. Here the authors show that biological specimens amassing <100 kDa can be resolved to better than 3 Å resolution using conventional defocus-based single-particle analysis methods.

• الناشر: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• موضوع: 147/143 / 631/1647/245/2160 / 631/45/535 / 631/535/1258/1259 / Alcohol dehydrogenase / Alcohol Dehydrogenase - chemistry / Alcohols / Cryoelectron Microscopy - methods / Crystallography, X-Ray / Cyclic AMP-Dependent Protein Kinases - chemistry / Electron microscopes / Electron microscopy / High resolution / Humanities and Social Sciences / Instrumentation / Ligands / Macromolecular Substances - chemistry / Macromolecules / Methemoglobin / Microscopy, Electron, Transmission - methods / Models, Molecular / Molecular Conformation / Molecular Weight / multidisciplinary / Phase plates / Plates (structural members) / Proteins / Proteins - chemistry / Science / Science (multidisciplinary) / Transmission electron microscopy