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- PDB-6r93: Cryo-EM structure of NCP-6-4PP -

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Entry
Database: PDB / ID: 6r93
TitleCryo-EM structure of NCP-6-4PP
Components
  • (Human alpha-satellite DNA (145- ...) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1
  • Histone H4
KeywordsDNA / DNA damage / Nucleosome / 6-4 photoproduct
Function / homology
Function and homology information


Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / Senescence-Associated Secretory Phenotype (SASP) / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / PKMTs methylate histone lysines / HDMs demethylate histones / RMTs methylate histone arginines / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Packaging Of Telomere Ends ...Condensation of Prophase Chromosomes / Oxidative Stress Induced Senescence / Senescence-Associated Secretory Phenotype (SASP) / DNA Damage/Telomere Stress Induced Senescence / HDACs deacetylate histones / PKMTs methylate histone lysines / HDMs demethylate histones / RMTs methylate histone arginines / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Packaging Of Telomere Ends / Interleukin-7 signaling / Meiotic synapsis / Cleavage of the damaged purine / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged pyrimidine / PRC2 methylates histones and DNA / HATs acetylate histones / Chromatin modifying enzymes / Formation of the beta-catenin:TCF transactivating complex / Metalloprotease DUBs / Factors involved in megakaryocyte development and platelet production / Amyloid fiber formation / Meiotic recombination / Estrogen-dependent gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / E3 ubiquitin ligases ubiquitinate target proteins / RNA Polymerase I Promoter Escape / RNA Polymerase I Promoter Opening / G2/M DNA damage checkpoint / Deposition of new CENPA-containing nucleosomes at the centromere / Processing of DNA double-strand break ends / Nonhomologous End-Joining (NHEJ) / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Pre-NOTCH Transcription and Translation / Ub-specific processing proteases / DNA methylation / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / NoRC negatively regulates rRNA expression / SUMOylation of chromatin organization proteins / UCH proteinases / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / 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 / DNA replication-dependent nucleosome assembly / telomere organization / innate immune response in mucosa / nuclear nucleosome / 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 / regulation of gene silencing / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / lipopolysaccharide binding / protein heterotetramerization / chromatin organization / double-strand break repair via nonhomologous end joining / antibacterial humoral response / nuclear chromosome, telomeric region / killing of cells of other organism / antimicrobial humoral immune response mediated by antimicrobial peptide / nuclear chromatin / protein ubiquitination / cadherin binding / blood coagulation / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / protein domain specific binding / cellular protein metabolic process / protein heterodimerization activity / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / nucleoplasm / extracellular region / nucleus / cytosol
CENP-T/Histone H4, histone fold / Histone H3/CENP-A / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / Histone H2B / Histone H2A, C-terminal domain / Histone H4, conserved site ...CENP-T/Histone H4, histone fold / Histone H3/CENP-A / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / Histone H2B / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone H2A conserved site / Histone H3 signature 2. / Histone H2B signature. / Histone H3 signature 1. / Histone H4 signature. / Histone H2A signature. / C-terminus of histone H2A / Centromere kinetochore component CENP-T histone fold / Core histone H2A/H2B/H3/H4
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsMatsumoto, S. / Cavadini, S. / Bunker, R.D. / Thoma, N.H.
Funding supportSwitzerland , Japan , 7件
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_160734/1Switzerland
European Union666068
European Commission667951
European Commission705354
Japan Society for the Promotion of ScienceJP18H05534Japan
Japan Society for the Promotion of ScienceJP16H06307Japan
Japan Agency for Medical Research and Development (AMED)JP18am0101076Japan
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 Report
SummaryFull reportAbout validation report
DateDeposition: Apr 2, 2019 / Release: Jun 12, 2019
RevisionDateData content typeProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release

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Structure visualization

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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
I: Human alpha-satellite DNA (145-MER)
J: Human alpha-satellite DNA (145-MER) with a 6-4PP at positions 95-96
A: Histone H3.1
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.1
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J


Theoretical massNumber of molelcules
Total (without water)201,92710
Polymers201,92710
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area57670 Å2
ΔGint-373 kcal/mol
Surface area74160 Å2
MethodPISA

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Components

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Human alpha-satellite DNA (145- ... , 2 types, 2 molecules IJ

#1: DNA chain Human alpha-satellite DNA (145-MER)


Mass: 44765.664 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: DNA chain Human alpha-satellite DNA (145-MER) with a 6-4PP at positions 95-96


Mass: 45038.852 Da / Num. of mol.: 1
Details: 145 bp human alpha-satellite with a 6-4 pyrimidine-pyrimidone (6-4PP) at base positions 95-96.
Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein/peptide , 4 types, 8 molecules AEBFCGDH

#3: Protein/peptide Histone H3.1 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone H3/i / Histone H3/j / Histone H3/k / Histone H3/l


Mass: 15719.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H3A, H3FA, HIST1H3B, H3FL, HIST1H3C, H3FC, HIST1H3D, H3FB, HIST1H3E, H3FD, HIST1H3F, H3FI, HIST1H3G, H3FH, HIST1H3H, H3FK, HIST1H3I, H3FF, HIST1H3J, H3FJ
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P68431
#4: Protein/peptide Histone H4 /


Mass: 11676.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Cell line (production host): JM109(DE3) / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#5: Protein/peptide Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 2
Details: ...GSHMSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04908
#6: Protein/peptide Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 14217.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BJ, H2BFR / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06899

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
16-4PP nucleosome1,2,3,4,5,60MULTIPLE SOURCES
2DNA1,21RECOMBINANT
3Histone H3.1, Histone H2A, Histone H2B3,5,61RECOMBINANT
4Histone H441RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: NO
Source (natural)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-ID
22
33
44
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33Escherichia coli BL21(DE3) (bacteria)469008
44Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.NameFormula
150 mMSodium chlorideNaClSodium chloride
250 mMHEPES
30.25 mMTCEP
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 0 mm / C2 aperture diameter: 50 µns / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: dev_3318: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION23D reconstruction
13REFMAC5.8.0238model refinementDNA only
14PHENIXdev-3318model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98378 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
14ZUXI
24ZUXJ
35Y0CA
45Y0CB
55Y0CC
65Y0CD
75Y0CE
85Y0CF
95Y0CG
105Y0CH
114.0E+54B
123EI4A

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