(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 …
(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 …
(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 …
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 …
(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 …
(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 …
Western blot quantification for Swd1 Loop 1 mutants.
(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 …
(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 …
Western blot quantification for Set1 RxxxRR helix mutants.
(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 …
(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 …
Western blot quantification for Swd1 Ubiquitin-binding mutants.
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 |
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 |