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- EMDB-4693: Structure of a human nucleosome at 3.5 A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-4693
TitleStructure of a human nucleosome at 3.5 A resolution
Map dataMasked Structure of a human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA at 3.5 A resolution.
Sample
  • Complex: human nucleosome particle
    • Complex: octamer of human histones
    • Complex: 171bp widom-601 DNA
Function / homology
Function and homology information


nucleosomal DNA binding / RNA polymerase II core promoter sequence-specific DNA binding / negative regulation of megakaryocyte differentiation / 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 / RNA polymerase II core promoter sequence-specific DNA binding / negative regulation of megakaryocyte differentiation / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / telomere organization / RNA Polymerase I Promoter Opening / SUMOylation of chromatin organization proteins / Assembly of the ORC complex at the origin of replication / DNA methylation / Condensation of Prophase Chromosomes / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / innate immune response in mucosa / PRC2 methylates histones and DNA / Defective pyroptosis / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / G2/M DNA damage checkpoint / B-WICH complex positively regulates rRNA expression / HDMs demethylate histones / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / Factors involved in megakaryocyte development and platelet production / Processing of DNA double-strand break ends / HATs acetylate histones / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / positive regulation of cell growth / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / RNA polymerase II cis-regulatory region sequence-specific DNA binding / Amyloid fiber formation / protein heterodimerization activity / enzyme binding / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / identical protein binding / nucleus / cytosol
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 H2A / Histone 2A / Histone H4, conserved site ...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 / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWilson MD / Nans A / Costa A
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
The Francis Crick InstituteFC0010065 United Kingdom
The Francis Crick InstituteFC0010061 United Kingdom
Citation
Journal: 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.
#1: Journal: Nat Commun / Year: 2019
Title: Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamerDNA looping and sliding along the histone octamer
Authors: Wilson MD / Renault L / Makell DP / Ghoneim M / Pye VE / Cherepanov P / Nans A / Rueda D / Cherepanov P / Costa A
History
DepositionMar 12, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4693.png

Downloads & links

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Map

FileDownload / File: emd_4693.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked Structure of a human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA at 3.5 A resolution.
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.20108458 - 0.42855772
Average (Standard dev.)0.0007975448 (±0.010560773)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 279.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z279.040279.040279.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2010.4290.001

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Supplemental data

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Mask #1

Fileemd_4693_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmasked Structure of a human nucleosome wrapped with...

Fileemd_4693_additional.map
AnnotationUnmasked Structure of a human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unmasked Structure of a human nucleosome wrapped with...

Fileemd_4693_additional_1.map
AnnotationUnmasked Structure of a human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 from relion-2.1b refinement of human...

Fileemd_4693_half_map_1.map
Annotationhalf map 2 from relion-2.1b refinement of human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1 from relion-2.1b refinement of human...

Fileemd_4693_half_map_2.map
Annotationhalf map 1 from relion-2.1b refinement of human nucleosome wrapped with 171bp of Widom-601 strong positioning DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human nucleosome particle

EntireName: human nucleosome particle
Components
  • Complex: human nucleosome particle
    • Complex: octamer of human histones
    • Complex: 171bp widom-601 DNA

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Supramolecule #1: human nucleosome particle

SupramoleculeName: human nucleosome particle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: recombinant human nucleosmes generated by expression of individual human histones and PCR synthesis of 171bp Widonm 601 DNA
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-DE3 / Recombinant plasmid: PET28a
Molecular weightTheoretical: 215 KDa

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Supramolecule #2: octamer of human histones

SupramoleculeName: octamer of human histones / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: recombinantly expressed human histones, H2A.1, H2B, H3.1, H4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-DE3 / Recombinant plasmid: PET28a

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Supramolecule #3: 171bp widom-601 DNA

SupramoleculeName: 171bp widom-601 DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1 / Details: widom-601 DNA generated by PCR
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.180 mg/mL
BufferpH: 7
Component:
ConcentrationName
10.0 mMTris
1.0 mMEDTAEthylenediaminetetraacetic acid
1.0 mMDTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.00039 kPa / Details: 40mA on EMS glowdischarge unit
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blotforce -1 blottime 4s.
Detailsquantified based on DNA absorbtion at A260

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 128000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Detailsgrids screened manully and loaded into krios, optimal grid selected for
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1300 / Average exposure time: 60.0 sec. / Average electron dose: 31.3 e/Å2 / Details: Falcon III counting mode 30 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 205680
Details: autopicked based on mnaully picked template in relion-2.1beta
CTF correctionSoftware - Name: Gctf (ver. 1.05) / Details: Full CTF correction
Startup modelType of model: OTHER
Details: model generated from on ab initio cryosparc 3D class
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1 beta)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.1 beta)
Details: 3d classification with three classes, two clases high resolution pooled for subsequent 3d refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1 beta)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1 beta) / Number images used: 123123
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3lz0

RefinementProtocol: RIGID BODY FIT

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