A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation
A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation
复制标题
DOI：
10.1016/j.jsb.2005.12.013
复制
发表时间：
2006-05-01
影响因子：
3
通讯作者：
Spahn, CMT
中科院分区：
文献类型：
--
作者：
Penczek, PA;Frank, J;Spahn, CMT
基于bootstrap的方法计算的三维方差cryo-EM地图重建从他们的投影集被施加到一个数据集的功能性核糖体复合物含有大肠杆菌70 S核糖体，tRNA，和延伸因子G（EF-G）。方差图显示了核糖体亚基间空间的高变异性区域：在tRNA的位置，在EF-G的推定位置，以及在L1蛋白附近。这一结果表明数据集的异质性。提出了一种聚焦分类的方法，将投影数据分成近似同质的子集。该方法是基于一个区域的识别和定位的高方差，随后的分类步骤调用通过使用一个3D球形掩模集中。初始分类后，创建模板体积，随后使用多参考3D投影对齐程序进行细化。在应用于核糖体数据集时，这两种结构被解释为来自具有结合的EF-G和空A位点的核糖体复合物，或者来自没有结合EF-G但具有被tRNA占据的A和P位点的复合物。所提出的聚焦分类方法被证明是一个成功的工具，在异构冷冻EM数据集的分析。密度图内相关性的相关计算证实了复合物的构象变异性，这可以用核糖体延伸周期来解释。(c)2006年爱思唯尔公司All rights reserved.
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 call 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. (c) 2006 Elsevier Inc. All rights reserved.