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- EMDB-3674: RNF169 chromatin binding region bound to a ubiquitylated nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-3674
TitleRNF169 chromatin binding region bound to a ubiquitylated nucleosome
Map dataMIU-LRM region of RNF169 bound to a ubiquitylated nucleosome core particle
Sample
  • Complex: RNF169/NCP-ub complex
    • Complex: RNF169 MIU-LRM region
    • Complex: H2AK13 ubiquitylated Nucleosome core particle
Biological speciesHomo sapiens (human) / Drosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.85 Å
AuthorsWilson MD / Kitevski-LeBlanc J / Durocher D / Rubinstein JL / Kay LE
CitationJournal: Elife / Year: 2017
Title: The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.
Authors: Julianne Kitevski-LeBlanc / Amélie Fradet-Turcotte / Predrag Kukic / Marcus D Wilson / Guillem Portella / Tairan Yuwen / Stephanie Panier / Shili Duan / Marella D Canny / Hugo van Ingen / ...Authors: Julianne Kitevski-LeBlanc / Amélie Fradet-Turcotte / Predrag Kukic / Marcus D Wilson / Guillem Portella / Tairan Yuwen / Stephanie Panier / Shili Duan / Marella D Canny / Hugo van Ingen / Cheryl H Arrowsmith / John L Rubinstein / Michele Vendruscolo / Daniel Durocher / Lewis E Kay /
Abstract: Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, ...Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes.
History
DepositionApr 17, 2017-
Header (metadata) releaseApr 26, 2017-
Map releaseMay 24, 2017-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3674.png

Downloads & links

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Map

FileDownload / File: emd_3674.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMIU-LRM region of RNF169 bound to a ubiquitylated nucleosome core particle
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.45 Å/pix.
x 128 pix.
= 185.6 Å		1.45 Å/pix.
x 128 pix.
= 185.6 Å		1.45 Å/pix.
x 128 pix.
= 185.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.028542077 - 0.15155181
Average (Standard dev.)0.003448616 (±0.015225172)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0290.1520.003

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Supplemental data

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Additional map: H2A K13 disulphide conjugated nuclesome core particlce

Fileemd_3674_additional.map
AnnotationH2A K13 disulphide conjugated nuclesome core particlce
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RNF169/NCP-ub complex

EntireName: RNF169/NCP-ub complex
Components
  • Complex: RNF169/NCP-ub complex
    • Complex: RNF169 MIU-LRM region
    • Complex: H2AK13 ubiquitylated Nucleosome core particle

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Supramolecule #1: RNF169/NCP-ub complex

SupramoleculeName: RNF169/NCP-ub complex / type: complex / ID: 1 / Parent: 0
Details: MIU-LRM region of RNF169 bound to H2AK13 disulphide ubiquitylated Nucleosome core particle
Molecular weightTheoretical: 230 kDa/nm

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Supramolecule #2: RNF169 MIU-LRM region

SupramoleculeName: RNF169 MIU-LRM region / type: complex / ID: 2 / Parent: 1
Details: SUMO-RNF169 residues 662-708 was expressed recombinantly including a DAAA helix stabilizing extension. SUMO tag was removed by Ulp1 cleavage prior to formation of RNF169/NCP-ub complex
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10 kDa/nm

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Supramolecule #3: H2AK13 ubiquitylated Nucleosome core particle

SupramoleculeName: H2AK13 ubiquitylated Nucleosome core particle / type: complex / ID: 3 / Parent: 1
Details: recombinant Drosophila histones wrapped with synthetic strong positioning Widom-601 DNA. Ubiquitin G76C covalently tethered to engineered K13C residue in H2A, prior to NCP reconstitution
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 220 kDa/nm

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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

Concentration0.57 mg/mL
BufferpH: 6.8 / Details: 10 mM tris pH 6.8, 30 mM KCl 13 and 1mM EDTA
GridModel: homemade / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
DetailsNCP-ub and RNF169 fragment were mixed at 1:5 ratio

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Electron microscopy

MicroscopeFEI TECNAI F20
Details5 separate sessions of manual data collection were combined.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 5 / Number real images: 867 / Average exposure time: 15.0 sec. / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 301275
CTF correctionSoftware - Name: CTFFIND (ver. 1.4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kx5


Details: .mrc file was geenrated in chimera. Model was low pass filtered to 50 angstrom
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 31760
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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