Structural basis for COMPASS recognition of an H2B-ubiquitinated nucleosome

  1. Evan J Worden
  2. Xiangbin Zhang
  3. Cynthia Wolberger  Is a corresponding author
  1. Johns Hopkins University School of Medicine, United States
6 figures, 2 tables and 1 additional file

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 …

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 …

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 …

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 …

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
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 …

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
Magnification81,000
Voltage (kV)300
Electron exposure (e–/Å2)50
Defocus range (μm)−1.0 to −2.5
Pixel size (Å)1.08
Symmetry imposedC1
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
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Saccharomyces cerevisiae)Yeast strain BY4741(Baker Brachmann et al., 1998)
AntibodyAnti-Histone H3 (mono-methyl K4) polyclonalAbcamAbcam Cat# ab8895, RRID:AB_306847WB (1:500)
AntibodyAnti-Histone H3 (di-methyl K4) polyclonalAbcamAbcam Cat# ab7766, RRID:AB_2560996WB (1:500)
AntibodyAnti-Histone H3 (tri-methyl K4) polyclonalAbcamAbcam Cat# ab8580, RRID:AB_306649WB (1:500)
AntibodyAnti-GAPDH monoclonalAbcamAbcam Cat# ab125247, RRID:AB_11129118WB (1:2000)
Recombinant DNA reagentpEW106This StudypQE-81L: H3 K4M
Recombinant DNA reagentpEW66This StudyCOMPASS expression plasmid pBIG1a containing Bre2, Swd1, Swd2, Sdc1 and Sgh1
Recombinant DNA reagentpEW107This StudyCOMPASS expression plasmid pBIG1b containing 6xHis-3xFLAG-Set1(762–1080), Swd3 and twin-strep-Spp1.
Recombinant DNA reagentpEW108This StudyCOMPASS expression plasmid pBIG1ab containing all COMPASS subunits.
Recombinant DNA reagentWT Set1This StudypEW111 pRS415: WT Set1
Recombinant DNA reagentWT Swd1This StudypEW113
pRS415: WT Swd1
Recombinant DNA reagentSet1(R936A)This StudypEW118
pRS415: Set1(R936A)
Recombinant DNA reagentSet1(R936E)This StudypEW120
pRS415: Set1(R936E)
Recombinant DNA reagentSet1(R901A)This StudypEW123
pRS415: Set1(R901A)
Recombinant DNA reagentSet1(R904A)This StudypEW124
pRS415: Set1(R904A)
Recombinant DNA reagentSet1(R908A)This StudypEW125
pRS415: Set1(R908A)
Recombinant DNA reagentSet1(R909A)This StudypEW126
pRS415: Set1(R909A)
Recombinant DNA reagentSet1(R901A, R904A, R908A, R909A)This StudypEW127
pRS415: Set1(R901A, R904A, R908A, R909A)
Recombinant DNA reagentSet1(R901E)This StudypEW128
pRS415: Set1(R901E)
Recombinant DNA reagentSet1(R904E)This StudypEW129
pRS415: Set1(R904E)
Recombinant DNA reagentSet1(R908E)This StudypEW130
pRS415: Set1(R908E)
Recombinant DNA reagentSet1(R909E)This StudypEW131
pRS415: Set1(R909E)
Recombinant DNA reagentSet1(R901E, R904E, R908E, R909E)This StudypEW132
pRS415: Set1(R901E, R904E, R908E, R909E)
Recombinant DNA reagentSwd1(L12A)This StudypEW139
pRS415: Swd1(L12A)
Recombinant DNA reagentSwd1(E14R)This StudypEW140
pRS415: Swd1(E14R)
Recombinant DNA reagentSwd1(E14A)This StudypEW141
pRS415: Swd1(E14A)
Recombinant DNA reagentSwd1(F9A, L12A, E14A)This StudypEW142
pRS415: Swd1(F9A, L12A, E14A)
Recombinant DNA reagentSwd1(E397R)This StudypEW144
pRS415: Swd1(E397R)
Recombinant DNA reagentSwd1(V263A)This StudypEW145
pRS415: Swd1(V263A)
Recombinant DNA reagentSwd1(I264A)This StudypEW146
pRS415: Swd1(I264A)
Recombinant DNA reagentSwd1(N265A)This StudypEW147
pRS415: Swd1(N265A)
Recombinant DNA reagentSwd1(K266A)This StudypEW148
pRS415: Swd1(K266A)
Commercial assay or kitMTase-GloPromegaV7601

Additional files

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