A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation - PubMed (original) (raw)
Review
. 2006 May;154(2):184-94.
doi: 10.1016/j.jsb.2005.12.013. Epub 2006 Feb 17.
Affiliations
- PMID: 16520062
- DOI: 10.1016/j.jsb.2005.12.013
Review
A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation
Pawel A Penczek et al. J Struct Biol. 2006 May.
Abstract
The bootstrap-based method for calculation of the 3D variance in cryo-EM maps reconstructed from sets of their projections was applied to a dataset of functional ribosomal complexes containing the Escherichia coli 70S ribosome, tRNAs, and elongation factor G (EF-G). The variance map revealed regions of high variability in the intersubunit space of the ribosome: in the locations of tRNAs, in the putative location of EF-G, and in the vicinity of the L1 protein. This result indicated heterogeneity of the dataset. A method of focused classification was put forward in order to sort out the projection data into approximately homogenous subsets. The method is based on the identification and localization of a region of high variance that a subsequent classification step can be focused on by the use of a 3D spherical mask. After initial classification, template volumes are created and are subsequently refined using a multireference 3D projection alignment procedure. In the application to the ribosome dataset, the two resulting structures were interpreted as resulting from ribosomal complexes with bound EF-G and an empty A site, or, alternatively, from complexes that had no EF-G bound but had both A and P sites occupied by tRNA. The proposed method of focused classification proved to be a successful tool in the analysis of the heterogeneous cryo-EM dataset. The associated calculation of the correlations within the density map confirmed the conformational variability of the complex, which could be interpreted in terms of the ribosomal elongation cycle.
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