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- EMDB-4767: Cryo-EM structure of NCP-6-4PP -

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Basic information

Entry
Database: EMDB / ID: EMD-4767
TitleCryo-EM structure of NCP-6-4PP
Map data
Sample6-4PP nucleosome
  • DNA
  • Histone H3.1, Histone H2A, Histone H2BHistone H3
  • (Histone H4) x 2
  • (nucleic-acidNucleic acid) x 2
  • Histone H3.1Histone H3
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
Function / homology
Function and homology information


negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / negative regulation of tumor necrosis factor-mediated signaling pathway / DNA replication-independent nucleosome assembly / telomere capping / interleukin-7-mediated signaling pathway / chromatin silencing / telomere organization / DNA replication-dependent nucleosome assembly / innate immune response in mucosa ...negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / negative regulation of tumor necrosis factor-mediated signaling pathway / DNA replication-independent nucleosome assembly / telomere capping / interleukin-7-mediated signaling pathway / chromatin silencing / telomere organization / DNA replication-dependent nucleosome assembly / innate immune response in mucosa / nuclear nucleosome / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / regulation of gene silencing / nuclear chromosome / DNA-templated transcription, initiation / regulation of megakaryocyte differentiation / nucleosome assembly / lipopolysaccharide binding / nucleosome / double-strand break repair via nonhomologous end joining / chromatin organization / nuclear chromosome, telomeric region / killing of cells of other organism / antibacterial humoral response / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / protein ubiquitination / cadherin binding / protein heterodimerization activity / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / nuclear chromatin / protein domain specific binding / cellular protein metabolic process / host cell nucleus / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / nucleus / cytosol
Histone H2A / CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H4 / Histone H2B / Histone H3/CENP-A
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsMatsumoto S / Cavadini S / Bunker RD / Thoma NH
Funding support Switzerland, Japan, 7 items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_160734/1 Switzerland
European Commission667951
European Union666068
Japan Society for the Promotion of ScienceJP18H05534 Japan
European Commission705354
Japan Agency for Medical Research and Development (AMED)JP18am0101076 Japan
Japan Society for the Promotion of ScienceJP16H06307 Japan
CitationJournal: Nature / Year: 2019
Title: DNA damage detection in nucleosomes involves DNA register shifting.
Authors: Syota Matsumoto / Simone Cavadini / Richard D Bunker / Ralph S Grand / Alessandro Potenza / Julius Rabl / Junpei Yamamoto / Andreas D Schenk / Dirk Schübeler / Shigenori Iwai / Kaoru ...Authors: Syota Matsumoto / Simone Cavadini / Richard D Bunker / Ralph S Grand / Alessandro Potenza / Julius Rabl / Junpei Yamamoto / Andreas D Schenk / Dirk Schübeler / Shigenori Iwai / Kaoru Sugasawa / Hitoshi Kurumizaka / Nicolas H Thomä /
Abstract: Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced ...Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
Validation ReportPDB-ID: 6r93

SummaryFull reportAbout validation report
History
DepositionApr 2, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseJun 12, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6r93
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4767.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 300 pix.
= 258. Å
0.86 Å/pix.
x 300 pix.
= 258. Å
0.86 Å/pix.
x 300 pix.
= 258. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.5
Minimum - Maximum-9.714905 - 13.968496
Average (Standard dev.)0.00073272863 (±0.44168124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 258.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z258.000258.000258.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-9.71513.9680.001

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Supplemental data

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Sample components

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Entire 6-4PP nucleosome

EntireName: 6-4PP nucleosome / Number of components: 10

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Component #1: protein, 6-4PP nucleosome

ProteinName: 6-4PP nucleosome / Recombinant expression: No
MassTheoretical: 199 kDa

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Component #2: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: synthetic construct (others)

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Component #3: protein, Histone H3.1, Histone H2A, Histone H2B

ProteinName: Histone H3.1, Histone H2A, Histone H2BHistone H3 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, Histone H4

ProteinName: Histone H4 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: nucleic-acid, Human alpha-satellite DNA (145-MER)

nucleic acidName: Human alpha-satellite DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG) ...Sequence:
(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC)(DA) (DA)(DC)(DC)(DA)(DA)(DA)(DA)(DG)(DG)(DC) (DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT)(DG) (DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT)(DT) (DT)(DT)(DG)(DA)(DT)(DG)(DG)(DA)(DG)(DC) (DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA)(DA) (DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DA) (DT)(DT)(DG)(DA)(DT)
MassTheoretical: 44.765664 kDa
SourceSpecies: Homo sapiens (human)

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Component #6: nucleic-acid, Human alpha-satellite DNA (145-MER) with a 6-4PP at...

nucleic acidName: Human alpha-satellite DNA (145-MER) with a 6-4PP at positions 95-96
Class: DNA
Details: 145 bp human alpha-satellite with a 6-4 pyrimidine-pyrimidone (6-4PP) at base positions 95-96.
Structure: OTHER / Synthetic: No
Sequence: (DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG) ...Sequence:
(DA)(DT)(DC)(DA)(DA)(DT)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DA)(DT) (DT)(DC)(DT)(DA)(DC)(DC)(DA)(DA)(DA)(DA) (DG)(DT)(DG)(DT)(DA)(DT)(DT)(DT)(DG)(DG) (DA)(DA)(DA)(DC)(DT)(DG)(DC)(DT)(DC)(DC) (DA)(DT)(DC)(DA)(DA)(DA)(DA)(DG)(DG)(DC) (DA)(DT)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DA)(DA)(DC)(DC)(DA)(DG)(DC)(DT)(DG) (DA)(DA)(DC)(DA)(DT)(DG)(DC)(DC)(DT)(DT) (DT)(DT)(DG)(DA)(T64)(DG)(DG)(DA)(DG)(DC) (DA)(DG)(DT)(DT)(DT)(DC)(DC)(DA)(DA)(DA) (DT)(DA)(DC)(DA)(DC)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DG)(DA)(DA)(DT)(DC)(DT)(DG) (DC)(DA)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DA) (DT)(DT)(DG)(DA)(DT)
MassTheoretical: 45.038852 kDa
SourceSpecies: Homo sapiens (human)

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Component #7: protein, Histone H3.1

ProteinName: Histone H3.1Histone H3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 15.719445 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #8: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.676703 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #9: protein, Histone H2A type 1-B/E

ProteinName: Histone H2A type 1-B/E
Details: GSHMSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.447825 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #10: protein, Histone H2B type 1-J

ProteinName: Histone H2B type 1-J / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.217516 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL / pH: 7.4
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 85 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 130000.0 X (nominal) / Cs: 0 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 98378
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Cross-correlation coefficient / Refinement space: REAL
Input PDB model: 4ZUX, 4ZUX, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 5Y0C, 4E54, 3EI4
Chain ID: I, J, A, B, C, D, E, F, G, H, B, A
Output model

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