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- EMDB-4762: Cryo-EM structure of NCP-6-4PP(-1)-UV-DDB -

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Basic information

Entry
Database: EMDB / ID: EMD-4762
TitleCryo-EM structure of NCP-6-4PP(-1)-UV-DDB
Map data
SampleUV-DDB bound to a 6-4PP containing nucleosome:
(histones) x 2 / DNA damage-binding proteins / DNA / Histone H3.1 / (Histone H4) x 2 / Histone H2A type 1-B/E / Histone H2B type 1-J / (nucleic-acidNucleic acid) x 2 / (DNA damage-binding protein ...) x 2
Function / homology
Function and homology information


Cleavage of the damaged purine / Recognition of DNA damage by PCNA-containing replication complex / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected purine / Meiotic synapsis / Interleukin-7 signaling / Packaging Of Telomere Ends / Pre-NOTCH Transcription and Translation / Formation of the beta-catenin:TCF transactivating complex ...Cleavage of the damaged purine / Recognition of DNA damage by PCNA-containing replication complex / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected purine / Meiotic synapsis / Interleukin-7 signaling / Packaging Of Telomere Ends / Pre-NOTCH Transcription and Translation / Formation of the beta-catenin:TCF transactivating complex / PRC2 methylates histones and DNA / Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / HDMs demethylate histones / PKMTs methylate histone lysines / Senescence-Associated Secretory Phenotype (SASP) / HATs acetylate histones / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / G2/M DNA damage checkpoint / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / UCH proteinases / Ub-specific processing proteases / TP53 Regulates Transcription of DNA Repair Genes / Gap-filling DNA repair synthesis and ligation in TC-NER / Metalloprotease DUBs / Dual incision in TC-NER / Formation of Incision Complex in GG-NER / RMTs methylate histone arginines / Nonhomologous End-Joining (NHEJ) / Processing of DNA double-strand break ends / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / DNA Damage Recognition in GG-NER / Formation of TC-NER Pre-Incision Complex / Deposition of new CENPA-containing nucleosomes at the centromere / RNA Polymerase I Promoter Opening / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation by small RNAs / Neddylation / Chromatin modifying enzymes / SIRT1 negatively regulates rRNA expression / Factors involved in megakaryocyte development and platelet production / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Amyloid fiber formation / Meiotic recombination / RNA Polymerase I Promoter Escape / Estrogen-dependent gene expression / NoRC negatively regulates rRNA expression / SUMOylation of chromatin organization proteins / B-WICH complex positively regulates rRNA expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / DNA methylation / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / Dual Incision in GG-NER / positive regulation by virus of viral protein levels in host cell / Cul4B-RING E3 ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / UV-damage excision repair / positive regulation of viral release from host cell / histone H2A monoubiquitination / WD40-repeat domain binding / positive regulation of gluconeogenesis / interaction with symbiont / response to UV-B / Cul4-RING E3 ubiquitin ligase complex / cullin family protein binding / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / interleukin-7-mediated signaling pathway / positive regulation of viral genome replication / regulation of mitotic cell cycle phase transition / protein autoubiquitination / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / response to UV / nuclear nucleosome / pyrimidine dimer repair / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / nucleosomal DNA binding / DNA-templated transcription, initiation / regulation of gene silencing by miRNA / nuclear chromosome / nucleotide-excision repair, DNA damage recognition / nucleotide-excision repair, DNA duplex unwinding / regulation of gene silencing / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / proteasomal protein catabolic process / regulation of megakaryocyte differentiation / nucleotide-excision repair / nucleotide-excision repair, DNA incision, 5'-to lesion
CPSF A subunit region / Histone H2B signature. / Trp-Asp (WD) repeats signature. / Histone H3 signature 2. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Histone H4 signature. / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H2A signature. ...CPSF A subunit region / Histone H2B signature. / Trp-Asp (WD) repeats signature. / Histone H3 signature 2. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Histone H4 signature. / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H2A signature. / WD40 repeat, conserved site / WD domain, G-beta repeat / Core histone H2A/H2B/H3/H4 / WD40-repeat-containing domain superfamily / CENP-T/Histone H4, histone fold / DNA damage-binding protein 2 / Histone H2A conserved site / Histone H2A, C-terminal domain / DNA damage-binding protein 1 / Histone H4, conserved site / WD40-repeat-containing domain / C-terminus of histone H2A / WD40/YVTN repeat-like-containing domain superfamily / Histone-fold / Histone H2A/H2B/H3 / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / WD40 repeat / Histone H2B / Histone H3/CENP-A
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1 / DNA damage-binding protein 1 / DNA damage-binding protein 2
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsMatsumoto S / Cavadini S / Bunker RD / Thoma NH
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 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: 6r8y

SummaryFull reportAbout validation report
DateDeposition: Apr 2, 2019 / Header (metadata) release: May 22, 2019 / Map release: Jun 12, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6r8y
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4762.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: 3.4 / Movie #1: 3.4
Minimum - Maximum-14.282897999999999 - 32.150463000000002
Average (Standard dev.)0.00045621197 (±0.9515588)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-14.28332.1500.000

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Supplemental data

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Sample components

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Entire UV-DDB bound to a 6-4PP containing nucleosome

EntireName: UV-DDB bound to a 6-4PP containing nucleosome / Number of components: 14

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Component #1: protein, UV-DDB bound to a 6-4PP containing nucleosome

ProteinName: UV-DDB bound to a 6-4PP containing nucleosome / Recombinant expression: No
MassTheoretical: 199 kDa

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Component #2: protein, histones

ProteinName: histones / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, DNA damage-binding proteins

ProteinName: DNA damage-binding proteins / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #4: protein, DNA

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

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Component #5: protein, histones

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

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Component #6: protein, Histone H3.1

ProteinName: Histone H3.1 / 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 #7: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.676703 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: protein, Histone H2A type 1-B/E

ProteinName: Histone H2A type 1-B/E / 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 #9: 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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Component #10: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.394426 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #11: 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)(DC)(DT)(DC)(DA) (DA)(DT)(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: 44.780672 kDa
SourceSpecies: Homo sapiens (human)

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Component #12: 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)(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)(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: 44.709645 kDa
SourceSpecies: Homo sapiens (human)

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Component #13: protein, DNA damage-binding protein 1

ProteinName: DNA damage-binding protein 1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 127.425812 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #14: protein, DNA damage-binding protein 2

ProteinName: DNA damage-binding protein 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 48.261277 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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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: 84000
3D reconstructionSoftware: RELION / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: 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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