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

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Basic information

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


negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / 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 ...negative regulation of tumor necrosis factor-mediated signaling pathway / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / 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 / nucleosomal DNA binding / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / telomere organization / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / epigenetic regulation of gene expression / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / innate immune response in mucosa / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / heterochromatin formation / PKMTs methylate histone lysines / Metalloprotease DUBs / Meiotic recombination / Pre-NOTCH Transcription and Translation / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / antimicrobial humoral immune response mediated by antimicrobial peptide / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / antibacterial humoral response / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / Processing of DNA double-strand break ends / gene expression / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / defense response to Gram-negative bacterium / Estrogen-dependent gene expression / killing of cells of another organism / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / cadherin binding / protein heterodimerization activity / Amyloid fiber formation / negative regulation of cell population proliferation / protein-containing complex / DNA binding / RNA binding / extracellular space / extracellular exosome / extracellular region / nucleoplasm / membrane / 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 H4, conserved site ...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 H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T 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 / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / 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: 4 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]
Revision 1.4May 22, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Superimposition on EM map
  • EMDB-4767
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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
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
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 / Source method: obtained synthetically
Details: 145 bp human alpha-satellite with a 6-4 pyrimidine-pyrimidone (6-4PP) at base positions 95-96.
Source: (synth.) Homo sapiens (human)

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Protein , 4 types, 8 molecules AEBFCGDH

#3: Protein Histone H3.1 / 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
Details: GSHMSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK
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
16-4PP nucleosomeCOMPLEXall0MULTIPLE SOURCES
2DNACOMPLEX#1-#21RECOMBINANT
3Histone H3.1, Histone H2A, Histone H2BCOMPLEX#3, #5-#61RECOMBINANT
4Histone H4COMPLEX#41RECOMBINANT
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)32630
33Escherichia coli BL21(DE3) (bacteria)469008
44Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium chlorideNaCl1
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 FIELD / 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
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: Cross-correlation coefficient
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
14ZUX

4zux

I4ZUX1
24ZUX

4zux

J4ZUX1
35Y0C

5y0c

A5Y0C2
45Y0C

5y0c

B5Y0C2
55Y0C

5y0c

C5Y0C2
65Y0C

5y0c

D5Y0C2
75Y0C

5y0c

E5Y0C2
85Y0C

5y0c

F5Y0C2
95Y0C

5y0c

G5Y0C2
105Y0C

5y0c

H5Y0C2
114.0E+54B4.0E+543
123EI4

3ei4

A3EI44

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