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Cryo-EM of macromolecular assemblies at near-atomic resolution

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.

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Figure 1: The cryo-EM density map reconstruction process.
Figure 2: Cα backbone model generation.
Figure 3: Atomic model generation.
Figure 4: CTF curve.
Figure 5: Mm-cpn refinement.
Figure 6: FSC curve.
Figure 7: SSE identification.
Figure 8: SSE correspondence.
Figure 9: Modeling in Gorgon.
Figure 10: Optimization with Coot.

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Acknowledgements

This research was supported by grants from the National Institutes of Health through the Nanomedicine Development Center (PN1EY016525), the Nanobiology Training Program (R90DK71504), the Institute of General Medical Sciences (R01GM079429, R01GM080139), the National Center for Research Resources (P41RR002250) and the National Science Foundation (IIS-0705644, IIS-0705474).

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Contributions

M.L.B. developed Gorgon, the cryo-EM–based modeling protocol and modeled Mm-cpn. S.J.L. developed EMAN and the reconstruction protocol. J.Z. performed the image processing and reconstructions for Mm-cpn. All authors contributed to the preparation of the paper.

Corresponding author

Correspondence to Wah Chiu.

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The authors declare no competing financial interests.

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Baker, M., Zhang, J., Ludtke, S. et al. Cryo-EM of macromolecular assemblies at near-atomic resolution. Nat Protoc 5, 1697–1708 (2010). https://doi.org/10.1038/nprot.2010.126

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