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- PDB-6r0c: Human-D02 Nucleosome Core Particle with biotin-streptavidin label -

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Basic information

Entry
Database: PDB / ID: 6r0c
TitleHuman-D02 Nucleosome Core Particle with biotin-streptavidin label
Components
  • (DNA (142-MER)) x 2
  • Histone H2A type 1
  • Histone H2B type 1-C/E/F/G/I
  • Histone H3.3
  • Histone H4
KeywordsDNA BINDING PROTEIN / chromatin / nucleosome / retrovirus
Function / homology
Function and homology information


negative regulation of chromosome condensation / Barr body / regulation of centromere complex assembly / muscle cell differentiation / pericentric heterochromatin formation / inner kinetochore / oocyte maturation / nucleus organization / spermatid development / subtelomeric heterochromatin formation ...negative regulation of chromosome condensation / Barr body / regulation of centromere complex assembly / muscle cell differentiation / pericentric heterochromatin formation / inner kinetochore / oocyte maturation / nucleus organization / spermatid development / subtelomeric heterochromatin formation / single fertilization / negative regulation of megakaryocyte differentiation / RNA polymerase II core promoter sequence-specific DNA binding / protein localization to CENP-A containing chromatin / 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 / embryo implantation / Meiotic synapsis / RNA Polymerase I Promoter Opening / 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 / 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 / multicellular organism growth / 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 / osteoblast differentiation / male gonad development / 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 / Senescence-Associated Secretory Phenotype (SASP) / positive regulation of cell growth / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / cell population proliferation / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / protein heterodimerization activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / Amyloid fiber formation / enzyme binding / protein-containing complex / DNA binding / RNA binding / extracellular space / extracellular exosome / extracellular region / nucleoplasm / identical protein binding / membrane
Similarity search - Function
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 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
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1 / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsPye, V.E. / Wilson, M.D. / Cherepanov, P. / Costa, A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
The Francis Crick InstituteFC0010061 United Kingdom
The Francis Crick InstituteFC0010065 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer.
Authors: Marcus D Wilson / Ludovic Renault / Daniel P Maskell / Mohamed Ghoneim / Valerie E Pye / Andrea Nans / David S Rueda / Peter Cherepanov / Alessandro Costa /
Abstract: Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase ...Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers.
History
DepositionMar 12, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.2May 15, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._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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Assembly

Deposited unit
A: Histone H3.3
B: Histone H4
C: Histone H2A type 1
D: Histone H2B type 1-C/E/F/G/I
E: Histone H3.3
F: Histone H4
G: Histone H2A type 1
H: Histone H2B type 1-C/E/F/G/I
I: DNA (142-MER)
J: DNA (142-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,18211
Polymers199,12810
Non-polymers551
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: fluorescence resonance energy transfer, single molecule FRET with fluorophores on DNA and protein, native gel electrophoresis, to assess proper wrapping and positioning of nucleosome, gel ...Evidence: fluorescence resonance energy transfer, single molecule FRET with fluorophores on DNA and protein, native gel electrophoresis, to assess proper wrapping and positioning of nucleosome, gel filtration, plus biochemical assays to assert activity on substrate
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area51850 Å2
ΔGint-392 kcal/mol
Surface area77490 Å2
MethodPISA

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3.3


Mass: 15360.983 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H3F3A, H3.3A, H3F3, PP781, H3F3B, H3.3B / Production host: Escherichia coli (E. coli) / References: UniProt: P84243
#2: Protein Histone H4


Mass: 11394.426 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
Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1 / H2A.1 / Histone H2A/ptl


Mass: 14121.537 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H2AG, H2AFP, HIST1H2AI, H2AFC, HIST1H2AK, H2AFD, HIST1H2AL, H2AFI, HIST1H2AM, H2AFN
Production host: Escherichia coli (E. coli) / References: UniProt: P0C0S8
#4: Protein Histone H2B type 1-C/E/F/G/I / Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone ...Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone H2B.k / H2B/k / Histone H2B.l / H2B/l


Mass: 13937.213 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H2BC, H2BFL, HIST1H2BE, H2BFH, HIST1H2BF, H2BFG, HIST1H2BG, H2BFA, HIST1H2BI, H2BFK
Production host: Escherichia coli (E. coli) / References: UniProt: P62807

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (142-MER)


Mass: 44427.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (142-MER)


Mass: 45071.785 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Human-D02 Nucleosome Core Particle with biotin-streptavidin labelCOMPLEXhuman histones refolded as an octamer with native human D02 sequence with flexible linker biotin.tetravalent streptavidin added onto refolded nucleosomes and sample crosslinked with glutaraldehyde#1-#60RECOMBINANT
2HistonesCOMPLEXHistones#1-#41RECOMBINANT
3DNACOMPLEXDNA#5-#61RECOMBINANT
Molecular weightValue: 0.286 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris1
21 mMEDTA1
320 mMsodium chlorideNaCl1
41 MMDTT1
SpecimenConc.: 0.176 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Streptavidin incubated D02-biotin nucleosomes were crosslinked with glutaraldehye. This was quenched and the sample spin concentrated
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 3.5 nm / Nominal defocus min: 1.5 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 28.3 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4182

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Processing

SoftwareName: PHENIX / Version: 1.14_3211: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4Gctf1.08CTF correction
7Cootmodel fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3712 / Details: obvious micrographs with cubic ice were discarded
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62196 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: The initial model was placed in the density using Chimera. Manual building was performed in Coot and final refinement was carried out using phenix.real_space_refine. Additional restraints ...Details: The initial model was placed in the density using Chimera. Manual building was performed in Coot and final refinement was carried out using phenix.real_space_refine. Additional restraints describing protein secondary structure, DNA base pairing and stacking were used in Phenix.
Atomic model buildingPDB-ID: 3UTB

3utb


Accession code: 3UTB / Source name: PDB / Type: experimental model

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