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- PDB-6t79: Structure of a human nucleosome at 3.2 A resolution -

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Entry
Database: PDB / ID: 6t79
TitleStructure of a human nucleosome at 3.2 A resolution
Components
  • (DNA (147-MER)) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-K
  • Histone H3.2
  • Histone H4
KeywordsNUCLEAR PROTEIN / Nucleosome / DNA / histones
Function / homology
Function and homology information


negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / interleukin-7-mediated signaling pathway / chromatin silencing / telomere organization / DNA replication-dependent nucleosome assembly / nucleosome => GO:0000786 / innate immune response in mucosa ...negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / interleukin-7-mediated signaling pathway / chromatin silencing / telomere organization / DNA replication-dependent nucleosome assembly / nucleosome => GO:0000786 / innate immune response in mucosa / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / nuclear chromosome / DNA-templated transcription, initiation / regulation of megakaryocyte differentiation / nucleosome assembly / nucleosome / double-strand break repair via nonhomologous end joining / chromosome, telomeric region => GO:0000781 / chromatin organization / killing of cells of other organism / antibacterial humoral response / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / protein ubiquitination / defense response to Gram-negative bacterium / protein heterodimerization activity / defense response to Gram-positive bacterium / chromatin => GO:0000785 / negative regulation of cell population proliferation / protein domain specific binding / cellular protein metabolic process / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / nucleus / cytosol
CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / Histone H2B ...CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / Histone H2B / Histone H3/CENP-A / Histone, subunit A / Histone, subunit A / Orthogonal Bundle / Mainly Alpha
Histone H2B type 1-K / Histone H2A type 1-B/E / Histone H4 / Histone H3.2
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsDodonova, S.O. / Zhu, F. / Dienemann, C. / Taipale, J. / Cramer, P.
Funding support Germany, 3items
OrganizationGrant numberCountry
European Research Council693023 Germany
European Molecular Biology OrganizationALTF-949-2016 Germany
Volkswagen Foundation Germany
CitationJournal: Nature / Year: 2020
Title: Nucleosome-bound SOX2 and SOX11 structures elucidate pioneer factor function.
Authors: Svetlana O Dodonova / Fangjie Zhu / Christian Dienemann / Jussi Taipale / Patrick Cramer /
Abstract: 'Pioneer' transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming. Pioneer factors can bind nucleosomal DNA to enable gene expression from regions ...'Pioneer' transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming. Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells. Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 21, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-K
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-K
I: DNA (147-MER)
J: DNA (147-MER)


Theoretical massNumber of molelcules
Total (without water)205,54910
Polymers205,54910
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58380 Å2
ΔGint-382 kcal/mol
Surface area74490 Å2
MethodPISA

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3.2 / Histone H3/m / Histone H3/o


Mass: 15389.036 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H3A, HIST2H3C, H3F2, H3FM, HIST2H3D / Plasmid: pET22b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus-RIL / References: UniProt: Q71DI3
#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
Plasmid: pET3a / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus-RIL / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 16707.277 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Plasmid: LIC-1B (MacroLabs) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus-RIL / References: UniProt: P04908
#4: Protein Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 13921.213 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BK, H2BFT, HIRIP1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus-RIL / References: UniProt: O60814

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (147-MER)


Mass: 45240.848 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (147-MER)


Mass: 45484.273 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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
1NucleosomeCOMPLEXNucleosome#1-#60MULTIPLE SOURCES
2ProteinsProteinCOMPLEXProteins#1-#41RECOMBINANT
3DNACOMPLEXDNA#5-#61RECOMBINANT
Molecular weightValue: 0.205 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
230 mMNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid1
42 mMDTT1
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 0.39 mB, 25 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K
Details: The sample was applied onto glow-discharged Quantifoil holey carbon grids. The grids were blotted from both sides for 5-10 seconds at 16*C in a chamber at 100% humidity and plunge-frozen ...Details: The sample was applied onto glow-discharged Quantifoil holey carbon grids. The grids were blotted from both sides for 5-10 seconds at 16*C in a chamber at 100% humidity and plunge-frozen into liquid ethane using a manual plunger.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: At least 50% of the data were collected at 25* stage tilt in order to partially compensate for preferred orientation of particles on the grid, and to improve angular distribution.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.125 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 Bioquantum / Energyfilter slit width: 30 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 368270 / Symmetry type: POINT
EM volume selectionDetails: 1733 vesicles and near-complete buds were picked from 61 tomograms. Subtomograms were extracted from the surface of the vesicles.
Num. of tomograms: 54 / Num. of volumes extracted: 2547
Atomic model buildingB value: 75 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6FQ5

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