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Nat Protoc. 2010 Sep;5(10):1697-708. doi: 10.1038/nprot.2010.126. Epub 2010 Sep 30.

Cryo-EM of macromolecular assemblies at near-atomic resolution.

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1
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.

Abstract

With single-particle electron cryomicroscopy (cryo-EM), it is possible to visualize large, macromolecular assemblies in near-native states. Although subnanometer resolutions have been routinely achieved for many specimens, state of the art cryo-EM has pushed to near-atomic (3.3-4.6 Å) resolutions. At these resolutions, it is now possible to construct reliable atomic models directly from the cryo-EM density map. In this study, we describe our recently developed protocols for performing the three-dimensional reconstruction and modeling of Mm-cpn, a group II chaperonin, determined to 4.3 Å resolution. This protocol, utilizing the software tools EMAN, Gorgon and Coot, can be adapted for use with nearly all specimens imaged with cryo-EM that target beyond 5 Å resolution. Additionally, the feature recognition and computational modeling tools can be applied to any near-atomic resolution density maps, including those from X-ray crystallography.

PMID:
20885381
PMCID:
PMC3107675
DOI:
10.1038/nprot.2010.126
[Indexed for MEDLINE]
Free PMC Article
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