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Structure. 2016 Feb 2;24(2):319-28. doi: 10.1016/j.str.2015.12.006. Epub 2016 Jan 14.

2.9 Å Resolution Cryo-EM 3D Reconstruction of Close-Packed Virus Particles.

Author information

1
Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, West Lafayette, IN 47907, USA.
2
Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan.
3
Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, West Lafayette, IN 47907, USA. Electronic address: jiang12@purdue.edu.

Abstract

Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic resolution 3D reconstructions from close-packed particles. Multiple independent de novo initial models were constructed to determine and cross-validate the particle parameters. The particles with consistent views were further refined including not only Euler angles and center positions but also defocus, astigmatism, beam tilt, and overall and anisotropic magnification. We demonstrated this strategy with a 2.9 Å resolution reconstruction of a 1.67 MDa virus-like particle of a circovirus, PCV2, recorded on 86 photographic films. The map resolution was further validated with a phase-randomization test and local resolution assessment, and the atomic model was validated with MolProbity and EMRinger. Close-packed virus particles were thus shown not only to be useful for high-resolution 3D reconstructions but also to allow data collection at significantly improved throughput for near-atomic resolution reconstructions.

KEYWORDS:

consensus criterion; cross-validation; de novo initial model; gold-standard FSC; single particle cryo-EM; true FSC

PMID:
26777413
PMCID:
PMC4740195
DOI:
10.1016/j.str.2015.12.006
[Indexed for MEDLINE]
Free PMC Article

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