Tutorial for high-throughput cryo-EM structure determination of amyloids: Dataset 2
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ABSTRACT:
SUBMITTER: Sjors H W Scheres
PROVIDER: EMPIAR-10943 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The formation of amyloid filaments is characteristic of various degenerative diseases. Recent breakthroughs in electron cryo-microscopy (cryo-EM) have led to atomic structure determination of multiple amyloid filaments, both of filaments assembled in vitro from recombinant proteins, and of filaments extracted from diseased tissue. These observations revealed that a single protein may adopt multiple different amyloid folds, and that in vitro assembly does not necessarily lead to the same filaments as those observed in disease. In order to develop relevant model systems for disease, and ultimately to better understand the molecular mechanisms of disease, it will be important to determine which factors determine the formation of distinct amyloid folds. High-throughput cryo-EM, in which structure determination becomes a tool rather than a project in itself, will facilitate the screening of large numbers of in vitro assembly conditions. To this end, we describe a new filament picking algorithm based on the Topaz approach, and we outline image processing strategies in Relion that enable atomic structure determination of amyloids within days.
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Project description: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.
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