Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome
- Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 4 supplements
Architecture of the COMPASS H2B-Ub nucleosome complex.
(a) Cryo-EM structure of the COMPASS-nucleosome complex. The unsharpened EM density showing two COMPASS molecules bound to the nucleosome is depicted as a semitransparent surface. The sharpened EM …
Figure 1—figure supplement 1
Analysis of COMPASS activty and binding to the nucleosome.
(a) Electrophoretic mobility shift assay (EMSA) showing COMPASS binding to different Nucleosome (NCP) variants. (b) Quantification of the EMSA shown in panel a. The binding interaction between …
Figure 1—figure supplement 2
Cryo-EM processing pipeline.
Figure 1—figure supplement 3
Cryo-EM map and model validation and example density.
(a) Fourier Shell Correlation (FSC) for the COMPASS-nucleosome structure. (b) Model-Map FSC curves calculated from the refined atomic coordinates and the full-map used for refinement (Full) and the …
Figure 1—figure supplement 4
Detailed views of EM density in the structure.
In all panels the sharpened EM density is shown as a semi-transparent gray surface. (a) Superimposition of Spp1 from PDB: 6BX3 and Spp1 from this study. The fragmented EM density at the end of Spp1 …
Figure 2
COMPASS makes multiple contacts with nucleosome DNA.
(a) Model of the COMPASS-nucleosome structure shown in cartoon representation and colored as in Figure 1. (b–d) Detailed views of COMPASS interactions with nucleosomal DNA. In all views the …
Figure 3 with 1 supplement
A conserved loop in Swd1 contacts the histone core.
(a) Close up view of the contact between Swd1 and the histone core. Swd1 is shown as a blue cartoon and the two loops that contact the histone core are colored red. (b) Detailed view of the specific …
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Figure 3—source data 1
Western blot quantification for Swd1 Loop 1 mutants.
- https://cdn.elifesciences.org/articles/53199/elife-53199-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Multiple sequence alignments of Swd1, Set1 and Bre2.
(a–c) Multiple sequence alignments were generated using the Clustle Omega software (Sievers et al., 2011). Species names are abbreviated (Sc = Saccharomyces cerevisiae, Sp = Schizosaccharomyces …
Figure 4
The RxxxRR helix binds to the H2A/H2B acidic patch.
(a) The COMPASS-nucleosome structure with COMPASS subunits surrounded by semi-transparent colored surfaces. The RxxxRR helix passes underneath the H2B-linked ubiquitin and is shown in cartoon …
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Figure 4—source data 1
Western blot quantification for Set1 RxxxRR helix mutants.
- https://cdn.elifesciences.org/articles/53199/elife-53199-fig4-data1-v2.xlsx
Figure 5
H2B-Ubiquitin recognition by different methyltransferases.
(a–c) Structures of H2B-ubiquitin activated methyltransferases bound to the H2B-Ub nucleosome. The structures are encompassed by a semitransparent gray surface, except for ubiquitin which is colored …
Figure 6 with 1 supplement
Structural basis for COMPASS recognition of H2B-ub.
(a) Model of the COMPASS H2B-Ub nucleosome complex. The unsharpened EM density is shown as a semi-transparent gray surface. The connecting density between Bre2 and ubiquitin is shown. (b) Detailed …
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Figure 6—source data 1
Western blot quantification for Swd1 Ubiquitin-binding mutants.
- https://cdn.elifesciences.org/articles/53199/elife-53199-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Comparison of Set1 catalytic domains from COMPASS in different states.
(a) Superimposition of the H2B-Ub nucleosome bound Set1 catalytic domain from S. cerevisiae (green) with the substrate bound, K. lactis Set1 catalytic domain from isolated COMPASS (PDB: 6CHG). (b) …
Tables
Table 1
Cryo-EM data collection, refinement and validation statistics
| Complex between COMPASS and the H2B-Ub nucleosome (EMDB-21157) (PDB 6VEN) | |
|---|---|
| Data collection and processing | |
| Magnification | 81,000 |
| Voltage (kV) | 300 |
| Electron exposure (e–/Å2) | 50 |
| Defocus range (μm) | −1.0 to −2.5 |
| Pixel size (Å) | 1.08 |
| Symmetry imposed | C1 |
| Initial particle images (no.) | 2,036,654 |
| Final particle images (no.) | 179,588 |
| Map resolution (Å) FSC threshold | 3.37 (0.143) |
| Map resolution range (Å) | 999–3.37 |
| Refinement | |
| Initial model used (PDB code) | PDB: 6NJ9, 6BX3, 6CHG |
| Model resolution (Å) FSC threshold | 3.40 (0.5) |
| Model resolution range (Å) | 47.6 to 3.40 |
| Map sharpening B factor (Å2) | −122.6 |
| Model composition Non-hydrogen atoms Protein residues Ligands | 24,032 2271 2 |
| B factors (Å2) Protein Ligand | 102.57 113.31 |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.004 0.696 |
| Validation MolProbity score Clashscore Poor rotamers (%) | 1.92 9.79 0.05 |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 94.01 5.99 0.0 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | Yeast strain BY4741 | (Baker Brachmann et al., 1998) | ||
| Antibody | Anti-Histone H3 (mono-methyl K4) polyclonal | Abcam | Abcam Cat# ab8895, RRID:AB_306847 | WB (1:500) |
| Antibody | Anti-Histone H3 (di-methyl K4) polyclonal | Abcam | Abcam Cat# ab7766, RRID:AB_2560996 | WB (1:500) |
| Antibody | Anti-Histone H3 (tri-methyl K4) polyclonal | Abcam | Abcam Cat# ab8580, RRID:AB_306649 | WB (1:500) |
| Antibody | Anti-GAPDH monoclonal | Abcam | Abcam Cat# ab125247, RRID:AB_11129118 | WB (1:2000) |
| Recombinant DNA reagent | pEW106 | This Study | pQE-81L: H3 K4M | |
| Recombinant DNA reagent | pEW66 | This Study | COMPASS expression plasmid pBIG1a containing Bre2, Swd1, Swd2, Sdc1 and Sgh1 | |
| Recombinant DNA reagent | pEW107 | This Study | COMPASS expression plasmid pBIG1b containing 6xHis-3xFLAG-Set1(762–1080), Swd3 and twin-strep-Spp1. | |
| Recombinant DNA reagent | pEW108 | This Study | COMPASS expression plasmid pBIG1ab containing all COMPASS subunits. | |
| Recombinant DNA reagent | WT Set1 | This Study | pEW111 pRS415: WT Set1 | |
| Recombinant DNA reagent | WT Swd1 | This Study | pEW113 pRS415: WT Swd1 | |
| Recombinant DNA reagent | Set1(R936A) | This Study | pEW118 pRS415: Set1(R936A) | |
| Recombinant DNA reagent | Set1(R936E) | This Study | pEW120 pRS415: Set1(R936E) | |
| Recombinant DNA reagent | Set1(R901A) | This Study | pEW123 pRS415: Set1(R901A) | |
| Recombinant DNA reagent | Set1(R904A) | This Study | pEW124 pRS415: Set1(R904A) | |
| Recombinant DNA reagent | Set1(R908A) | This Study | pEW125 pRS415: Set1(R908A) | |
| Recombinant DNA reagent | Set1(R909A) | This Study | pEW126 pRS415: Set1(R909A) | |
| Recombinant DNA reagent | Set1(R901A, R904A, R908A, R909A) | This Study | pEW127 pRS415: Set1(R901A, R904A, R908A, R909A) | |
| Recombinant DNA reagent | Set1(R901E) | This Study | pEW128 pRS415: Set1(R901E) | |
| Recombinant DNA reagent | Set1(R904E) | This Study | pEW129 pRS415: Set1(R904E) | |
| Recombinant DNA reagent | Set1(R908E) | This Study | pEW130 pRS415: Set1(R908E) | |
| Recombinant DNA reagent | Set1(R909E) | This Study | pEW131 pRS415: Set1(R909E) | |
| Recombinant DNA reagent | Set1(R901E, R904E, R908E, R909E) | This Study | pEW132 pRS415: Set1(R901E, R904E, R908E, R909E) | |
| Recombinant DNA reagent | Swd1(L12A) | This Study | pEW139 pRS415: Swd1(L12A) | |
| Recombinant DNA reagent | Swd1(E14R) | This Study | pEW140 pRS415: Swd1(E14R) | |
| Recombinant DNA reagent | Swd1(E14A) | This Study | pEW141 pRS415: Swd1(E14A) | |
| Recombinant DNA reagent | Swd1(F9A, L12A, E14A) | This Study | pEW142 pRS415: Swd1(F9A, L12A, E14A) | |
| Recombinant DNA reagent | Swd1(E397R) | This Study | pEW144 pRS415: Swd1(E397R) | |
| Recombinant DNA reagent | Swd1(V263A) | This Study | pEW145 pRS415: Swd1(V263A) | |
| Recombinant DNA reagent | Swd1(I264A) | This Study | pEW146 pRS415: Swd1(I264A) | |
| Recombinant DNA reagent | Swd1(N265A) | This Study | pEW147 pRS415: Swd1(N265A) | |
| Recombinant DNA reagent | Swd1(K266A) | This Study | pEW148 pRS415: Swd1(K266A) | |
| Commercial assay or kit | MTase-Glo | Promega | V7601 |
