(a) Front (top) and side (bottom) views of a composite cryo-EM reconstruction of human SIRT6-nucleosome. Maps from focused refinements of SIRT6 (magenta – Rossmann fold and Zn-finger domains …
The structures of human sirtuins are depicted – the Rossmann fold domain in blue, Zinc-finger in tan and the helix bundle between the two in green.
(a) Complete data processing scheme. Maps colored in rainbow represent the local resolution of the reconstructions. Hollow blue volume represents the mask used for focused classification and …
Complete data processing scheme. Maps colored in rainbow represent the local resolution of the reconstructions. FSC curves are depicted as a function of resolution in angstrom. CryoSPARC v.3 and v.4 …
(a) Close-up view of the SIRT6 Zn-finger interacting with the nucleosomal acidic patch. (b) View showing map details around the nucleosome dyad. (c) SIRT6 Rossmann fold domain interaction with the …
(a) Cryo-EM densities of the nucleotide base pairs at the dyad (yellow) and positions ± 4 (green) showing the orientation of the DNA in the SIRT6-nucleosome complex. Besides the dyad, the positions …
(a) UMAP projection of the latent embeddings of a subset of SIRT6-nucleosome particles representing the concerted movement of the Rossmann fold and the DNA terminus. (b) Structural representation of …
Particle images from CryoSPARC refinement were Fourier cropped to a size of 128x128 pixels. They were subjected to CryoDRGN analysis (run1). Ten representative volumes of the first Principal …
(a) Projection of per residue flexibility contribution calculated from molecular dynamics simulations of the nucleosome without SIRT6. The balls at the DNA residues are colored according to the …
(a) Close-up view on zinc-finger interactions with the acidic patch. Color code as in Figure 1. (b) Protein sequence alignment of human sirtuins. Red boxes and asterisks depict the residues of SIRT6 …
(a) Electrophoretic mobility shift assay to test binding of Widom-601 nucleosome to wt and mutant SIRT6. Lower and upper shifted bands visible in SIRT6 wt lane likely correspond to one or two SIRT6 …
(a) Top view of the SIRT6 bound nucleosome. Two distinct densities were observed between SIRT6 and the DNA. (b) Side view of the SIRT6 bound nucleosome. The third helix of SIRT6 (α3), which is part …
Z containing nucleosomes and canonical (H2A containing) ones. Used for creating Figure 3f.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Escherichia coli) | BL21-RIL | N/A | ||
Recombinant DNA reagent | SIRT6 (plasmid) | AddGene | #28271 | SIRT6 (PDB: 3PKJ) in pET28a-LIC |
Recombinant DNA reagent | Mutant SIRT6 (DNA fragment) | SynBio | K170A, R172A, R175A, R178A, R220A, R231A and R248A | |
Recombinant DNA reagent | Widom601 DNA (plasmid) | Dyer et al., 2004 | Gift from Dr. K. Mohideen-Abdul. | |
Recombinant DNA reagent | X. laevis H2A, H2B, H3, H4 histones (plasmids) | Luger et al., 1999; Dyer et al., 2004 | Gift from Dr. K. Mohideen-Abdul. | |
Recombinant DNA reagent | Human H2A.Z.1 (plasmid) | AddGene | #42629 | H2A.Z.1 in pET28a |
Antibody | H3K27ac (rabbit monoclonal) | CellSignaling | #8173 S | Diluted: 1:1000 |
Antibody | H3K9ac (rabbit polyclonal) | Abcam | Ab4441 | Diluted: 1:2500 |
Antibody | H3 (mouse monoclonal) | CellSignaling | #14269 S | Diluted: 1:1000 |
Software, algorithm | SerialEM | v4.1 | Automated data collection | |
Software, algorithm | EPU | Thermo Fisher | v3.4 | Automated data collection |
Software, algorithm | Warp | Warp | v1.0.9 | Micrograph preprocessing |
Software, algorithm | cryoSPARC | cryoSPARC | v4 | Image analysis |
Software, algorithm | RELION | RELION | v3.1 | Image analysis |
Software, algorithm | crYOLO | crYOLO | v1.9.6 | Particle picking |
Software, algorithm | cryoDRGN | cryoDRGN | v1.1 | Image analysis – structural flexibility analysis |
Software, algorithm | DeepEMhancer | DeepEMhancer | v0.8 | Map sharpening |
Software, algorithm | UCSF Chimera/ChimeraX | UCSF ChimeraX | v1.16/1.6 | Rigid body fitting, visualisation and figures |
Software, algorithm | Isolde | Isolde | v1.6 | Flexible fitting, model refinement |
Software, algorithm | PHENIX | PHENIX | v1.21 | Model refinement |
Software, algorithm | AMBER20 suit | AMBER20 | MD simulations |
Supplementary information for cryo-EM and molecular dynamics symulations.
(a) Cryo-EM data collection, refinement and validation statistics. (b) Frequency of conformational clusters for each MD ensemble. Clusters featuring the H3 tail protruding between the DNA and the octamer are marked by a star.