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- EMDB-2236: Negative Staining Structure of Human Polycomb Repressive Complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2236
TitleNegative Staining Structure of Human Polycomb Repressive Complex 2 bound to the co-factor AEBP2
Map dataNegative Staining Reconstruction of the Polycomb Repressive Complex 2 bound to the co-factor AEBP2
Sample
  • Sample: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
  • Protein or peptide: RbAp48RBBP4
  • Protein or peptide: Ezh2
  • Protein or peptide: Suz12
  • Protein or peptide: AEBP2
  • Protein or peptide: EED
KeywordsPolycomb / PRC2 / epigenetic / gene silencing / nucleosome / labeling / chemical cross-linking / chromatin
Function / homologyESC/E(Z) complex / SET domain
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsCiferri C / Lander GC / Maiolica A / Herzog F / Aebersold R / Nogales E
CitationJournal: Elife / Year: 2012
Title: Molecular architecture of human polycomb repressive complex 2.
Authors: Claudio Ciferri / Gabriel C Lander / Alessio Maiolica / Franz Herzog / Ruedi Aebersold / Eva Nogales /
Abstract: Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by ...Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. Here, we present the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, we have localized all the PRC2 subunits and their functional domains and generated a detailed map of interactions. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity.DOI:http://dx.doi.org/10.7554/eLife.00005.001.
History
DepositionNov 21, 2012-
Header (metadata) releaseDec 12, 2012-
Map releaseDec 12, 2012-
UpdateDec 12, 2012-
Current statusDec 12, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2236.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative Staining Reconstruction of the Polycomb Repressive Complex 2 bound to the co-factor AEBP2
Voxel sizeX=Y=Z: 3.14 Å
Density
Contour LevelBy AUTHOR: 3.55 / Movie #1: 3.55
Minimum - Maximum-6.36135721 - 22.46074295
Average (Standard dev.)0.0 (±0.99999964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 351.68002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.143.143.14
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z351.680351.680351.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-6.36122.4610.000

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Supplemental data

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Sample components

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Entire : Negative Staining Reconstruction of Polycomb repressive Complex 2...

EntireName: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
Components
  • Sample: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
  • Protein or peptide: RbAp48RBBP4
  • Protein or peptide: Ezh2
  • Protein or peptide: Suz12
  • Protein or peptide: AEBP2
  • Protein or peptide: EED

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Supramolecule #1000: Negative Staining Reconstruction of Polycomb repressive Complex 2...

SupramoleculeName: Negative Staining Reconstruction of Polycomb repressive Complex 2 bound to the co-factor AEBP2
type: sample / ID: 1000 / Oligomeric state: Pentameric Complex / Number unique components: 5
Molecular weightTheoretical: 265 MDa

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Macromolecule #1: RbAp48

MacromoleculeName: RbAp48 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFast Bac
SequenceGO: ESC/E(Z) complex / InterPro: SET domain

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Macromolecule #2: Ezh2

MacromoleculeName: Ezh2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFast Bac
SequenceGO: ESC/E(Z) complex / InterPro: SET domain

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Macromolecule #3: Suz12

MacromoleculeName: Suz12 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFast Bac
SequenceGO: ESC/E(Z) complex / InterPro: SET domain

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Macromolecule #4: AEBP2

MacromoleculeName: AEBP2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFast Bac
SequenceGO: ESC/E(Z) complex / InterPro: SET domain

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Macromolecule #5: EED

MacromoleculeName: EED / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Nucleus
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFast Bac
SequenceGO: ESC/E(Z) complex / InterPro: SET domain

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.6
Details: 25 mM Hepes pH 7.5, 138 mM NaCl, 10% Glycerol, 0.05% NP40, 1 mM Tris(2-carboxyethyl) phosphine hydrochloride [TCEP]
StainingType: NEGATIVE / Details: 2% Uranyl Formate
GridDetails: 400 mesh copper carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.2 mm / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 80000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder model: OTHER
TemperatureAverage: 298 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateApr 11, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 500 / Average electron dose: 20 e/Å2
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: whole micrograph
Final two d classificationNumber classes: 1000
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF
Software - Name: Appion, Leginon, Spider, Imagic, EMAN2, SPARX
Number images used: 39527
DetailsMaterial and Methods section of Paper

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
DetailsProtocol: local rigid-body fitting algorithm
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: Chimera
DetailsProtocol: local rigid-body fitting algorithm
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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