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 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.

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

“…Another option could be that, due to the negative charge of the oxygen which may result in attenuated positive potentials, its corresponding density is simply not seen [32][33][34][35][36]. Our observations are consistent with previously resolved cryo-EM maps derived from 200 keV electron microscopes [13], [14]. We expect that use of an energy filter should allow achievement of higher resolution, as demonstrated for the recent reconstruction of mouse apoferritin at 2.0 Å, where images were recorded with a higher frame rate detector and a more sophisticated 200 keV microscope [22].…”

“…Such sophisticated equipment is however expensive, requiring high-level strategies to maintain microscope stability and performance, so that the method would appear to be accessible in a daily manner to only few laboratories. Recent publications have explored the potential of 200 kV microscope to obtain reconstructions higher that 3 Å [13], [14] [15], [16] and [17], see Table S2. The dedicated instrument used to resolve those molecular structures is, again, highly sophisticated, and is in addition, costly.…”

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

“…Phase contrast methods such as defocusing the objective lens or the use of phase plates, especially the Volta phase plate (VPP) 7 , enable imaging of biological specimens with an acceptable (albeit still limiting) amount of radiation damage 8 . Despite the recent success in high-resolution reconstructions of small macromolecule proteins aided by a VPP including streptavidin (52 kDa tetramer, ~3.2 Å) 9 and human haemoglobin (64 kDa tetramer, ~3.2 Å) 10 or using conventional defocus-based data collection for methaemoglobin (64 kDa tetramer, ~2.8 Å and ~3.2 Å) and alcohol dehydrogenase (82 kDa dimer, ~2.7 Å) 11 , the routine analysis of such samples remains very challenging. To circumvent this problem, several protein engineering approaches have been employed to increase the size of particles.…”

Topaz-Denoise: general deep denoising models for cryoEM and cryoET

“…Conventional cryoEM methods for improving contrast in micrographs include downsampling, bandpass filtering, and Wiener filtering 2,3 . However, these methods do not address the specific noise properties of micrographs and often do not provide interpretable results, which is increasingly becoming an issue as researchers attempt to resolve small and non-globular proteins 4,5 .…”