Volume 20, Issue 2, Pages (February 2012)

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Volume 20, Issue 2, Pages 237-247 (February 2012) Iterative Stable Alignment and Clustering of 2D Transmission Electron Microscope Images Zhengfan Yang, Jia Fang, Johnathan Chittuluru, Francisco J. Asturias, Pawel A. Penczek Structure Volume 20, Issue 2, Pages 237-247 (February 2012) DOI: 10.1016/j.str.2011.12.007 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Flowchart of Stable Alignment and Clustering Only clusters comprising images with alignment parameters that are stable (at a given pixel error threshold) across several independent rounds of within-cluster alignment are retained. Images in unstable clusters are sent back to the unassigned image pool for reclustering. See also Figure S5. Structure 2012 20, 237-247DOI: (10.1016/j.str.2011.12.007) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Flowchart of Iterative Stable Alignment and Clustering The membership of clusters generated by four semi-independent SAC runs is compared, and only clusters with reproducible membership are retained. Images in clusters with low reproducibility are sent back to the unassigned image pool for reclustering. Structure 2012 20, 237-247DOI: (10.1016/j.str.2011.12.007) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 ISAC Results Obtained for the Data Set of EF-Tu Ribosomal Complex (A) Raw EM images. (B) Selection of ISAC cluster averages matched to projections of the X-ray structure. (C) Common lines volume compared with a map derived from the X-ray model. Scale bar corresponds to 10 nm. See also Figures S1–S3 and S6–S11. Structure 2012 20, 237-247DOI: (10.1016/j.str.2011.12.007) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 ISAC Analysis of hRNAPII EM Images (A) Raw EM images of hRNAPII. (B) A selection of hRNAPII ISAC cluster averages matched to projections of the X-ray structure of yeast RNAPII (pdb 1WCM). (C) A 3D map of hRNAPII derived by applying common lines to the ISAC averages shown in (B) (left), compared to a map of the homologous yeast RNAPII (right) derived from its X-ray structure. Scale bars correspond to 10 nm in (A and B) and 5 nm in (C). See also Figure S4. Structure 2012 20, 237-247DOI: (10.1016/j.str.2011.12.007) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Conformational Variability of hRNAPII Revealed by ISAC Cluster Averages (A) Selected hRNAPII ISAC averages showing changes in the position/appearance of the clamp domain (marked by yellow arrowheads). (B) hRNAPII volumes obtained after CD-PCA analysis of resampled ISAC averages show variability in clamp structure. (C) Two 3D maps obtained by competitive refinement of hRNAPII images using the volumes in (B) as initial references show alternative conformations of the hRNAPII clamp domain. Scale bars correspond to 10 nm in (A) and 5 nm in (B and C). Structure 2012 20, 237-247DOI: (10.1016/j.str.2011.12.007) Copyright © 2012 Elsevier Ltd Terms and Conditions