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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 H3.3H3F3A
  • Histone H4
KeywordsDNA BINDING PROTEIN / chromatin / nucleosome / retrovirus
Function / homology
Function and homology information


negative regulation of chromosome condensation / Barr body / subtelomeric heterochromatin assembly / regulation of centromere complex assembly / muscle cell differentiation / chromosome, subtelomeric region / pericentric heterochromatin assembly / nucleus organization / nucleosomal DNA binding / spermatid development ...negative regulation of chromosome condensation / Barr body / subtelomeric heterochromatin assembly / regulation of centromere complex assembly / muscle cell differentiation / chromosome, subtelomeric region / pericentric heterochromatin assembly / nucleus organization / nucleosomal DNA binding / spermatid development / negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / oogenesis / DNA replication-independent nucleosome assembly / telomere capping / single fertilization / chromatin silencing / RNA polymerase II core promoter sequence-specific DNA binding / telomere organization / DNA replication-dependent nucleosome assembly / innate immune response in mucosa / nuclear nucleosome / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / osteoblast differentiation / embryo implantation / nuclear chromosome / DNA-templated transcription, initiation / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / multicellular organism growth / cell population proliferation / male gonad development / double-strand break repair via nonhomologous end joining / chromatin organization / nuclear chromosome, telomeric region / positive regulation of cell growth / antibacterial humoral response / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / protein ubiquitination / RNA polymerase II cis-regulatory region sequence-specific DNA binding / protein heterodimerization activity / defense response to Gram-positive bacterium / nuclear chromatin / protein domain specific binding / cellular protein metabolic process / host cell nucleus / enzyme binding / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / identical protein binding / nucleus / cytosol
C-terminus of histone H2A / Histone-fold / Histone H3/CENP-A / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone H4, conserved site / Histone H2A, C-terminal domain ...C-terminus of histone H2A / Histone-fold / Histone H3/CENP-A / Histone H2B / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone H2A/H2B/H3 / Histone H4, conserved site / Histone H2A, C-terminal domain / Histone H2A conserved site / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / Centromere kinetochore component CENP-T histone fold
Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.3 / Histone H2A type 1
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.
Validation Report
SummaryFull reportAbout validation report
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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4692
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Water0
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
  • Download structure data
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 / H3F3A


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 / Manganese


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
2HistonesHistoneCOMPLEXHistones#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 mMEDTAEthylenediaminetetraacetic acid1
320 mMsodium chlorideNaClSodium chloride1
41 MMDTT1
SpecimenConc.: 0.176 mg/ml
Details: Streptavidin incubated D02-biotin nucleosomes were crosslinked with glutaraldehye. This was quenched and the sample spin concentrated
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELDBright-field microscopy / 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 / 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

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