Molecular architecture of the yeast Mediator complex
- Stanford University School of Medicine, United States
- University of California, San Francisco, United States
- Structural Bioinformatics Unit, France
Figures
Figure 1 with 2 supplements
Holoenzyme cross-linking and modeling results and methodology.
(A) Cross-links were identified by searching MS2 product ion spectra against concatenated target and decoy databases containing 52 Saccharomyces cerevisiae transcription proteins +520 sequence …
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Figure 1—source data 1
Categorization of cross-links with respect to module and data source.
Numbers of unique cross-linked residue pairs (‘cross-links’) and cross-linked spectral matches used for integrative modeling of Mediator apo-complex (‘Total Mediator’) as well as total number of holoenzyme cross-links identified in this study (‘Total [this study]’).
- https://doi.org/10.7554/eLife.08719.004
Figure 1—figure supplement 1
Purification of Native S. cerevisiae Holoenzyme complex.
(A) Schematic of the affinity capture and chromatographic fractionation of S. cerevisiae holoenzyme complex from whole-cell extract using a double-affinity tag and stepwise cleavage protocol. In the …
Figure 1—figure supplement 2
Annotated product ion spectra for selected cross-linked peptides.
HCD product ion spectra of BS3 cross-linked Mediator tryptic peptides classified by Protein Prospector. Spectra representing highly confident (SVM score 5.1) to less confident (SVM score 0.0) …
Figure 2 with 5 supplements
Mediator complex architecture.
(A) Mediator subunit localization density map colored by individual subunit. (B) Mediator localization density map (solid grey) calculated from the highest scoring model cluster and shown at a …
Figure 2—figure supplement 1
Schematic of integrative structure determination highlighting the individual data inputs and the four stages in our approach.
https://doi.org/10.7554/eLife.08719.008
Figure 2—figure supplement 2
Input Model Representations.
Individual Mediator subunit model representations are grouped according by membership within the Head, Middle, and Tail modules. For each subunit, a schematic representation of the model …
Figure 2—figure supplement 3
Cluster analysis of the solution ensemble.
(A) Number of satisfied intra-module cross-links plotted against the score for all 165,523 models. The vertical brown line represents the score threshold used to select the best scoring 500 models. …
Figure 2—figure supplement 4
Exhaustiveness of sampling and robustness of cross-link data.
(A) Comparison of localization density maps calculated from the ensemble of solutions (500 best-scoring models) for the entire sample of models, the first half, the second half, and for jackknifing …
Figure 2—figure supplement 5
Representation of subunit position precision.
(A) Root-mean-square fluctuation (RMSF) plots for each subunit of the Middle and Tail modules show how precisely the residues of all proteins were localized within the best cluster of models. For …
Figure 3
Mediator subunit domain interaction matrix.
Average domain–domain contact map calculated for the cluster of best-scoring solutions. Long sequences are divided into domains of 200 residues. The intensity of the color for each box is …
Figure 4
Architecture of the Mediator Middle module.
(A) Blow-out views of the Middle module subunit localization maps with the first view and coloring identical to Figure 2A and the second view related by a 180° rotation around the y-axis. (B) …
Figure 5
Architecture of the Mediator Tail module.
(A) Subunit localization within the Tail module. For each Tail subunit, the corresponding localization density is shown within a semi-transparent Tail density (grey) and colored according to Figure …
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Figure 5—source data 1
HHpred comparative modeling results for Tail module proteins.
- https://doi.org/10.7554/eLife.08719.016
Figure 6
Novel structural insights into Mediator complex architecture and module connectivity.
(A) Subunit localization density for Med17 (blue) reveals the extension of the unmodeled N-terminal domain to contact Middle module subunits. (Left A panel) The N-terminus of the Head subunit Med17 …
Figure 7 with 1 supplement
Holoenzyme cross-linking data indicate different conformational states between the holoenzyme and the core Mediator initiation complex.
(A) Docking of our Mediator model to the EM density of Plaschka et al. (2015) provides a subunit architectural map that is highly consistent with the 12 Mediator to pol II cross-links of Plaschka et …
Figure 7—figure supplement 1
Further validation of the Mediator model.
Agreement between the structural model of the Mediator complex and the data for the core initiation complex collected by Plaschka et al. (2015). (A) Comparison between our model (left), the EM map …
Additional files
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Supplementary file 1
Full list of Mediator–Mediator cross-linked peptides discovered by mass spectrometry.
Separate Microsoft Excel File.
- https://doi.org/10.7554/eLife.08719.020
