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- PDB-6r94: Cryo-EM structure of NCP_THF2(-3) -

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Basic information

Entry
Database: PDB / ID: 6r94
TitleCryo-EM structure of NCP_THF2(-3)
Components
  • (Human alpha-satellite DNA (145- ...) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1Histone H3
  • Histone H4
KeywordsDNA BINDING PROTEIN / DNA damage / Nucleosome / 6-4 photoproduct
Function / homology
Function and homology information


protein localization to CENP-A containing chromatin / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome / epigenetic regulation of gene expression / Packaging Of Telomere Ends / Chromatin modifying enzymes / negative regulation of tumor necrosis factor-mediated signaling pathway / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere ...protein localization to CENP-A containing chromatin / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome / epigenetic regulation of gene expression / Packaging Of Telomere Ends / Chromatin modifying enzymes / negative regulation of tumor necrosis factor-mediated signaling pathway / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / chromatin organization => GO:0006325 / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / Interleukin-7 signaling / telomere organization / RNA Polymerase I Promoter Opening / DNA replication-dependent chromatin assembly / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / chromatin organization => GO:0006325 / DNA methylation / innate immune response in mucosa / SIRT1 negatively regulates rRNA expression / Condensation of Prophase Chromosomes / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / PRC2 methylates histones and DNA / Defective pyroptosis / RNA Polymerase I Promoter Escape / HDACs deacetylate histones / nuclear chromosome / Nonhomologous End-Joining (NHEJ) / NoRC negatively regulates rRNA expression / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / B-WICH complex positively regulates rRNA expression / lipopolysaccharide binding / G2/M DNA damage checkpoint / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / Metalloprotease DUBs / DNA Damage/Telomere Stress Induced Senescence / RMTs methylate histone arginines / HDMs demethylate histones / nucleosome assembly / Meiotic recombination / Pre-NOTCH Transcription and Translation / PKMTs methylate histone lysines / HCMV Early Events / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / UCH proteinases / nucleosome / DNA-templated transcription initiation / E3 ubiquitin ligases ubiquitinate target proteins / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Processing of DNA double-strand break ends / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / Senescence-Associated Secretory Phenotype (SASP) / killing of cells of another organism / Oxidative Stress Induced Senescence / antibacterial humoral response / Estrogen-dependent gene expression / chromosome, telomeric region / antimicrobial humoral immune response mediated by antimicrobial peptide / Ub-specific processing proteases / cadherin binding / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / protein heterodimerization activity / Amyloid fiber formation / negative regulation of cell population proliferation / protein domain specific binding / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / extracellular region / membrane / nucleoplasm / nucleus / cytosol
Similarity search - Function
Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A ...Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Centromere kinetochore component CENP-T histone fold / CENP-T/Histone H4, histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA (> 100) / DNA (> 10) / DNA / Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMatsumoto, S. / Cavadini, S. / Bunker, R.D. / Thoma, N.H.
Funding support Switzerland, Japan, 7items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_160734/1 Switzerland
European Union666068
European Commission667951
European Commission705354
Japan Society for the Promotion of ScienceJP18H05534 Japan
Japan Society for the Promotion of ScienceJP16H06307 Japan
Japan Agency for Medical Research and Development (AMED)JP18am0101076 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.
History
DepositionApr 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Author supporting evidence / Data collection / Database references
Category: citation / em_image_scans / em_single_particle_entity
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
I: Human alpha-satellite DNA (145-MER)
J: Human alpha-satellite DNA (145-MER) with abasic sites at positions 97-98
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)201,33210
Polymers201,33210
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area60530 Å2
ΔGint-385 kcal/mol
Surface area77620 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: 44756.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: DNA chain Human alpha-satellite DNA (145-MER) with abasic sites at positions 97-98


Mass: 44452.434 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Protein , 4 types, 8 molecules AEBFCGDH

#3: Protein Histone H3.1 / Histone H3 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone ...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 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, ...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 Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 2
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 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
IDNameTypeEntity IDParent-IDSource
1THF2 (-3) containing nucleosomeCOMPLEXall0MULTIPLE SOURCES
2Human DNACOMPLEX#1-#21RECOMBINANT
3Histone H4COMPLEX#41RECOMBINANT
4Histone H3.1 / 2A / 2BCOMPLEX#3, #5-#61RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32360
33Escherichia coli (E. coli)562
44Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium chlorideNaClSodium chloride1
250 mMHEPES1
30.25 mMTCEP1
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 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder 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
9REFMAC5.8.0238model refinementDNA ONLY
10PHENIXdev-3318model refinement
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
14RELION23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78672 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14ZUXI1
24ZUXJ1
35Y0CA1
45Y0CB1
55Y0CC1
65Y0CD1
75Y0CE1
85Y0CF1
95Y0CG1
105Y0CH1
114.0E+54B1
123EI4A1

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