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. 2021 May 5;10:e65168. doi: 10.7554/eLife.65168

Figure 2. The Spc110pNCC binds near the N-terminus of Spc97p.

(A) Spc110p N-terminal region secondary structure prediction, showing lack of predicted secondary structure for the first 111 residues. Also shown are Spc110pCM1(117-146) and the Spc110pNCC(164-208) regions. (B) Structure of Xrcc4-Spc110p164-207, where Spc110pNCC residues 164–203 are resolved. (C) Spc110pNCC structure fit into γ-tubulin ring complex (γTuRC) cryo-EM density map (gray surface, EMDB ID 2799) along with γ-tubulin small complex (γTuSC) pseudo-atomic model (PDB ID 5FLZ) (Kollman et al., 2015; Greenberg et al., 2016). The majority of crosslinking mass spectrometry (XL-MS) distance restraints are satisfied by this model. Satisfied and violated disuccinimidyl suberate (DSS) crosslinks are shown in cyan and purple, respectively. Satisfied and violated 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinks are shown in blue and red, respectively. Crosslinks that are satisfied by either Spc110p monomer are shown twice, one for each monomer. (D) Localization density map for the ensemble of integrative models consisting of two adjacent γTuSCs, each bound to an Spc110p1-220 dimer. The map shows the positions of different parts of the complex in the ensemble of models from the top cluster; maps for all components are contoured at 2.5% of their respective maximum voxel values. The modeling results shown are based on the γTuSC-Spc110p1-220-GCN4 crosslinks; similar results were obtained using γTuSC-Spc110p1-401-GST crosslinks (see Appendix 1).

Figure 2.

Figure 2—figure supplement 1. Overview of crosslinking mass spectrometry (XL-MS) datasets.

Figure 2—figure supplement 1.

XL-MS datasets for γ-tubulin small complex (γTuSC) crosslinked to Spc110p1-220-GCN4 dimer (A, B) or Spc110p1-401-GST (C, D) with either EDC (A, C) or DSS (B, D). Each colored rectangle represents a protein in the crosslinked sample. The lengths of the rectangles are proportional to the number of residues in each protein. Crosslinks between γTuSC components and Spc110p1-111, Spc110pCM1(117-146), and Spc110pNCC (164-208) regions are color coded orange, green, and purple, respectively. (E) Table of crosslinks between Spc110pNCC(164-208) and γTuSC. Spc110p crosslinks marked with an asterisk are satisfied on either monomer within Spc110pNCC(164-208).
Figure 2—figure supplement 2. Results of integrative modeling of the Spc110p-γ-tubulin small complex (γTuSC) complex.

Figure 2—figure supplement 2.

The modeling results shown are based on the γTuSC-Spc110p1-220 GCN4 crosslinks; similar results were obtained in all cases using γTuSC-Spc110p1-401-GST crosslinks (see Appendix 1). (A) Monomer of Spc110p1-220-GCN4 bound to γTuSC. γTuSC and Spc110p NCC, which were kept fixed in the modeling, are shown as spherical beads with one bead per residue. Spc110p1-163 is shown as a localization density map, showing the positions of the Spc110p N-terminal domain (NTD) in the ensemble of models from the top cluster. (B) Dimer of Spc110p1-220-GCN4 bound to γTuSC, with a representation similar to (A). (C) A γTuSC monomer with EDC and DSS crosslinks from the NTD (residues 3–113) of the Spc110p1-220-GCN4 dimer construct mapped onto it (note that residues 3–113 in this construct correspond to residues 1–111 in Spc110). Each red sphere represents a residue in TuSC where the Spc110p1-111 crosslinks. The size of the red sphere indicates the number of residues in Spc110p1-111 that crosslink there. The smallest sphere indicates one residue from Spc110p1-111 crosslinks to that γTuSC residue; the largest spheres show where four or more residues from Spc110p1-111 crosslink. The red circle highlights a cluster of crosslink sites between Spc110p1-111 and Spc97p or Spc98p at the intra-γTuSC interface. (D) Dimer of Spc110p1-220-GCN4 bound to adjacent γTuSCs, shown as a localization density map, similar to Figure 1D. Maps for all components are contoured at 2.5% of their respective maximum voxel values. We highlight in purple a hypothetical path integrating our modeling and structural work showing how Spc110p bridges the inter-γTuSC interface and reaches to the adjacent γTuSC where it crosslinks to the N-terminal regions of Spc97p and Spc98p at the intra-γTuSC interface.
Figure 2—figure supplement 3. The four stages of integrative modeling of the Spc110-γ-tubulin small complex (γTuSC) complex.

Figure 2—figure supplement 3.

This schematic describes the integrative structure modeling procedures used in this paper. The first row details the information to be used in modeling. The background color of each information source indicates where the information is applied in modeling, as detailed in the key at the top. The second row describes how each information source is converted into spatial restraints. The third row details the sampling protocol. The last row details the analysis and validation steps of the modeling.
Figure 2—figure supplement 4. Results for sampling exhaustiveness protocol for modeling the complex of Spc110p1-220-GCN4 dimer with γ-tubulin small complex (γTuSC).

Figure 2—figure supplement 4.

(A) Results of test 1, convergence of the model score, for the 2840 good-scoring models; the scores do not continue to improve as more models are computed essentially independently. The error bar represents the standard deviations of the best scores, estimated by repeating sampling of models 10 times. The red dotted line indicates a lower bound reference on the total score. (B) Results of test 2, testing similarity of model score distributions between samples 1 (red) and 2 (blue); the difference in distribution of scores is not significant (Kolmogorov–Smirnov two-sample test p-value >0.05) and the magnitude of the difference is small (the Kolmogorov–Smirnov two-sample test statistic D is 0.05); thus, the two score distributions are effectively equal. (C) Results of test 3, three criteria for determining the sampling precision (Y-axis), evaluated as a function of the root-mean-square deviation (RMSD) clustering threshold (X-axis). First, the p-value is computed using the χ2-test for homogeneity of proportions (red dots). Second, an effect size for the χ2-test is quantified by the Cramer’s V value (blue squares). Third, the population of models in sufficiently large clusters (containing at least 10 models from each sample) is shown as green triangles. The vertical dotted gray line indicates the RMSD clustering threshold at which three conditions are satisfied (p-value>0.05 [dotted red line], Cramer’s V < 0.10 [dotted blue line], and the population of clustered models > 0.80 [dotted green line]), thus defining the sampling precision of 23.3 Å. (D) Populations of sample 1 and 2 models in the clusters obtained by threshold-based clustering using the RMSD threshold of 23.3 Å. Cluster precision is shown for each cluster. (E, F) Results of test 4: comparison of localization probability densities of models from sample As and B for the major cluster (98.1% population). The cross-correlation of the density maps of the two samples is 0.99 for the Spc110 maps (green).