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. 2019 Feb 14;8:e44239. doi: 10.7554/eLife.44239

Figure 2. Structure determination and model of the Ctf19c.

(A) Two-dimensional class averages showing various Ctf19c projections. (B) Initial Ctf19c density map with twofold symmetry applied. (C) Molecular model of the Ctf19c. The twofold symmetry axis is marked by an arrow. Subunits from one monomeric assembly are colored according to their identities. Those related by twofold symmetry are colored gray. The face view corresponds to the orientation shown in panel B.

Figure 2.

Figure 2—figure supplement 1. Initial three-dimensional cryo-EM reconstructions of the Ctf19c.

Figure 2—figure supplement 1.

(A) Data collection summary for the reconstruction show in in panel C. Images of crosslinked Ctf19c particles were collected manually at 200 kV. (B) Initial model used as a reference for three-dimensional classifications. The model was calculated from a small number of two-dimensional class averages using the program e2initialmodel.py (Tang et al., 2007). (C) Three-dimensional classification results for particles selected from images described in panel A. (D) Comparison of three-dimensional volumes calculated from uncrosslinked Ctf19c samples with or without Cnn1-Wip. With the exception of the missing density corresponding to Cnn1-Wip1, the two volumes showed nearly identical organization and features.
Figure 2—figure supplement 2. Data processing summary for Ctf19c structure determination.

Figure 2—figure supplement 2.

(A) Summary of data cleaning and pooling to generate an initial set of particles used for three-dimensional refinements in panel B. Three datasets were collected at 300 kV. The first two (left) were pooled immediately and the third (right) was added after initial three-dimensional classification. Images from the third dataset (right) contained mild crystalline ice contamination, and strict filtering substantially lowered the yield of useable particles relative to the total number. (B) Good particles were subjected to three-dimensional refinement and classification. Resolutions correspond to gold-standard FSC estimations (Scheres and Chen, 2012) and are provided as indicators of relative map quality. The central map at the bottom of the panel, which displays high-quality density throughout the particle volume, was used for molecular modeling and coordinate refinement. Ctf3c organization was determined with the help of the map shown to its left. The map at the right side of the panel shows high-resolution features that aided model building for the COMA complex.
Figure 2—figure supplement 3. Model summary and examples of map quality.

Figure 2—figure supplement 3.

(A) Fourier shell correlation (FSC) plots for half-maps (independent reconstructions, gold standard FSC; Scheres and Chen, 2012) and for the final Ctf19c model versus the final reconstruction. (B) Local resolution shown for the entire volume (left) and for a central slice through the volume (right) used for modeling. (C) An overview of the full Ctf19c structure with areas of interest labeled. (D) Examples of the density map with the corresponding Ctf19c model shown. Numbers correspond to those shown in panel C, and model colors correspond to those used in Figure 2C.