title: A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, 6431 Fannin, MSB 6.218, Houston, TX 77030, USA. Pawel.A.Penczek@uth.tmc.edu
authors.forename: Pawel A
authors.initials: PA
authors.lastname: Penczek
authors.forename: Joachim
authors.initials: J
authors.lastname: Frank
authors.forename: Christian M T
authors.initials: CM
authors.lastname: Spahn
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods / Q000706:statistics & numerical data; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D008958:Models, Molecular; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012343:RNA, Transfer