National Center for Macromolecular Imaging
Single Particle reconstruction (EMAN)
Dr Steve Ludtke


Single particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single particle analysis software package, which was first publicly distributed in 2000. We present a 3-dimensional (3-D) reconstruction of native naked GroEL to ~11.5 Å performed entirely with EMAN. We demonstrate that the single particle reconstruction, x-ray scattering data and x-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single particle reconstruction technique and x-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single particle and x-ray structures exhibits some small domain motions in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single particle reconstruction.

Selected Publications

 
  • Brink, J., Ludtke, S. J., Yang, C. Y., Gu, Z. W., Wakil, S. J. and Chiu, W. (2002). Quaternary structure of human fatty acid synthase by electron cryomicroscopy. Proc. Natl. Acad. Sci. U S A 99: 138-143.

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  • Ludtke, S.J., Jakana, J., Song, J.-L., Chuang, D. and Chiu, W. (2001). A 11.5 Å single particle reconstruction of GroEL using EMAN. J. Mol. Biol. 314: 253-262.

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  • Ludtke, S.J., Baldwin, P.R. and Chiu, W. (1999). EMAN: Semi-automated software for high resolution single particle reconstructions. J. Struct. Biol. 128: 82-97.

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  • Ludtke, S.J., and Chiu, W. (2002). Image restoration in sets of noisy electron micrographs. Proceedings of the IEEE International Symposium on Biomedical Imaging IEEE Cat 02EX608C., WA-SS-2.2.

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  • Ludtke, S.J. and Chiu, W. (2003). Focal pair merging for contrast enhancement of single particles. J Struct Biol, 144: 73-78.

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