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.
Highlights
Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons has been a longstanding goal of the cryo-electron microscopy community
We previously demonstrated that a transmission electron microscope (TEM) operating at 200 keV equipped with a K2 Summit direct electron detector (DED) could be used to resolve a ~150 kDa protein complex to ~2.6 Å using conventional defocusbased single-particle analysis (SPA) methods[12]
This prompted us to investigate whether high-resolution reconstructions of macromolecules
Summary
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. Determining ~3 Å resolution reconstructions of stable, conformationally and/or compositionally homogeneous specimens by SPA has become almost routine, with an increasing number of structures at ~2 Å resolution or better being reported[6,7,8] This resolution regime has expanded the potential of cryo-EM SPA for structure-based drug design, for targets that are less amenable to other structure determination techniques due to limited sample quantity or recalcitrance to crystallization. Due to the limited success in imaging smaller macromolecules by cryo-EM, the technique has primarily been used to visualize large complexes, with ~99% of all cryo-EM SPA reconstructions resolved to better than 5 Å resolution comprising macromolecules amassing >200 kDa. We previously demonstrated that a transmission electron microscope (TEM) operating at 200 keV equipped with a K2 Summit direct electron detector (DED) could be used to resolve a ~150 kDa protein complex to ~2.6 Å using conventional defocusbased SPA methods[12]. We provide the sub-nanometer single-particle cryo-EM structure of a sub-50 kDa macromolecular complex – the 43 kDa catalytic domain of protein kinase A
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