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- EMDB-3675: Nucleosome core particle Ubiquitylated at H2A Lys-13 -

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Basic information

Entry
Database: EMDB / ID: 3675
TitleNucleosome core particle Ubiquitylated at H2A Lys-13
Map dataNucleosome core particle with ubquitin covalently tethered to H2A lysine 13
SampleNCP-ub complex
  • H2AK13 ubiquitylated Nucleosome core particle
SourceDrosophila / / arthropod / image: Drosophila melanogaster
MethodCryo EM / single particle reconstruction / 8.8 Å resolution
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 / 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.
DateDeposition: Apr 17, 2017 / Header (metadata) release: May 3, 2017 / Map release: May 3, 2017 / Last update: Jul 12, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF CHIMERA
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF CHIMERA
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3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3675.map.gz (map file in CCP4 format, 8389 KB)
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.45 Å
Density
Contour Level:0.015 (by author), 0.015 (movie #1):
Minimum - Maximum-0.01582847 - 0.13095534
Average (Standard dev.)0.0036272337 (0.015028647)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
CellA=B=C: 185.6 Å
α=β=γ: 90 deg.

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
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0160.1310.004

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Supplemental data

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Sample components

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Entire NCP-ub complex

EntireName: NCP-ub complex
Details: H2AK13 disulphide ubiquitylated Nucleosome core particle
Number of components: 2

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Component #1: protein, NCP-ub complex

ProteinName: NCP-ub complex
Details: H2AK13 disulphide ubiquitylated Nucleosome core particle
Recombinant expression: No

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Component #2: protein, H2AK13 ubiquitylated Nucleosome core particle

ProteinName: H2AK13 ubiquitylated Nucleosome core particle
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
Recombinant expression: No
SourceSpecies: Drosophila / / arthropod / image: Drosophila melanogaster
Source (engineered)Expression System: Escherichia coli / / bacteria /

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: Cryo EM
Sample solutionSpecimen conc.: 0.65 mg/ml
Buffer solution: 10 mM Tris-Cl 17 pH 7.5, 50 mM KCl, 1 mM EDTA
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE MIXTURE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 36 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 25000 X (nominal), 34483 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 175

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 11063
3D reconstructionSoftware: RELION / Resolution: 8.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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