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- EMDB-8247: 53BP1 bound to a ubiquitylated and methylated nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-8247
Title53BP1 bound to a ubiquitylated and methylated nucleosome
Map dataNCP-ubme
Sample
  • Complex: NCP-ubme
    • Complex: NCP-ubme
      • Complex: Widom-601 DNA
      • Complex: H4Kc20me2
      • Complex: Histone H3
      • Complex: Histone H2A.1 K13RK36RT16S
      • Complex: Histone H2B.1
Biological speciessynthetic construct (others) / Xenopus laevis (African clawed frog) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.73 Å
AuthorsWilson MD / Benlekbir S / Sicheri F / Rubinstein JL / Durocher D
CitationJournal: Nature / Year: 2016
Title: The structural basis of modified nucleosome recognition by 53BP1.
Abstract: DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases ...DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.
History
DepositionJun 13, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8247.png

Downloads & links

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Map

FileDownload / File: emd_8247.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNCP-ubme
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.016747313 - 0.12817763
Average (Standard dev.)0.0026634254 (±0.014669847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 185.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z185.600185.600185.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0170.1280.003

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Supplemental data

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Sample components

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Entire : NCP-ubme

EntireName: NCP-ubme
Components
  • Complex: NCP-ubme
    • Complex: NCP-ubme
      • Complex: Widom-601 DNA
      • Complex: H4Kc20me2
      • Complex: Histone H3
      • Complex: Histone H2A.1 K13RK36RT16S
      • Complex: Histone H2B.1

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Supramolecule #1: NCP-ubme

SupramoleculeName: NCP-ubme / type: complex / ID: 1 / Parent: 0
Details: Recombinant ubiquitylated and methylated nucleosome core particle
Molecular weightTheoretical: 218 KDa

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Supramolecule #2: NCP-ubme

SupramoleculeName: NCP-ubme / type: complex / ID: 2 / Parent: 1
Details: Modified nuclesome core particles, H2A enzymatically ubiquitylated on H2A K15, H4 chemically alkylated at K20C to create dimethyl lysine analog
Molecular weightTheoretical: 226 KDa

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Supramolecule #3: Widom-601 DNA

SupramoleculeName: Widom-601 DNA / type: complex / ID: 3 / Parent: 2
Details: 145 bp fragment of Widom-601 strong nucleosome positioning DNA sequence, gift from Curt Davey (Vasudevan et. al, 2010, J.Mol.Biol.)
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pUC57-8x145bp
Molecular weightTheoretical: 882.31 KDa

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Supramolecule #4: H4Kc20me2

SupramoleculeName: H4Kc20me2 / type: complex / ID: 4 / Parent: 2
Details: Histone H4 alkylated at position 20 to create dimethyl lysine analog
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2349

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Supramolecule #5: Histone H3

SupramoleculeName: Histone H3 / type: complex / ID: 5 / Parent: 2
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD1874

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Supramolecule #6: Histone H2A.1 K13RK36RT16S

SupramoleculeName: Histone H2A.1 K13RK36RT16S / type: complex / ID: 6 / Parent: 2
Details: Histone H2A.1 covalently cross-linked at K15 to ubiquitin at G76 by an isopeptide bond
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2647

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Supramolecule #7: Histone H2B.1

SupramoleculeName: Histone H2B.1 / type: complex / ID: 7 / Parent: 2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2644

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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

Concentration0.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
45.0 mMKClpotassium chloride
0.5 mMEDTAEthylenediaminetetraacetic acid
1.0 mMDTT

Details: High concentration NCP-ubme/GST-53BP1 complex in 200 mM salt was diluted just prior to grid freezing.
GridModel: electron micrsocopy sciences / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 40.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: Plunged into liquid ethane-propane (FEI VITROBOT MARK III).

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 227 / Average exposure time: 0.5 sec. / Average electron dose: 36.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 52843
Details: Automatically picked from roughly 1000 particles using a manually picked template
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kx5


Details: Resized to the same pixel size as the data and high-pass filtered to 40 Angstrom resolution
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 10133
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsLocal fitting was performed in Chimera using rigid body fitting. No associate co-ordinates were deposited.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 1

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