Journal
NATURE PROTOCOLS
Volume 5, Issue 10, Pages 1697-1708Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2010.126
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Funding
- National Institutes of Health through Nanomedicine Development Center [PN1EY016525]
- Nanobiology Training Program [R90DK71504]
- Institute of General Medical Sciences [R01GM079429, R01GM080139]
- National Center for Research Resources [P41RR002250]
- National Science Foundation [IIS-0705644, IIS-0705474]
- NATIONAL CENTER FOR RESEARCH RESOURCES [P41RR002250] Funding Source: NIH RePORTER
- NATIONAL EYE INSTITUTE [PN1EY016525] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R90DK071504] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM079429, R01GM080139] Funding Source: NIH RePORTER
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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 angstrom) 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 angstrom 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 angstrom 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.
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