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

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Basic information

Entry
Database: EMDB / ID: EMD-3675
TitleNucleosome core particle Ubiquitylated at H2A Lys-13
Map dataNucleosome core particle with ubquitin covalently tethered to H2A lysine 13
Sample
  • Complex: NCP-ub complex
    • Complex: H2AK13 ubiquitylated Nucleosome core particle
Biological speciesDrosophila (fruit flies)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.8 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) releaseMay 3, 2017-
Map releaseMay 3, 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: 0.015
  • 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3675.png

Downloads & links

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Map

FileDownload / File: emd_3675.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNucleosome core particle with ubquitin covalently tethered to H2A lysine 13
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.01582847 - 0.13095534
Average (Standard dev.)0.0036272337 (±0.015028647)
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-0.0160.1310.004

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Supplemental data

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Sample components

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Entire : NCP-ub complex

EntireName: NCP-ub complex
Components
  • Complex: NCP-ub complex
    • Complex: H2AK13 ubiquitylated Nucleosome core particle

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Supramolecule #1: NCP-ub complex

SupramoleculeName: NCP-ub complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: H2AK13 disulphide ubiquitylated Nucleosome core particle

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Supramolecule #2: H2AK13 ubiquitylated Nucleosome core particle

SupramoleculeName: H2AK13 ubiquitylated Nucleosome core particle / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #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 (fruit flies)
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

Concentration0.65 mg/mL
BufferpH: 7.5 / Details: 10 mM Tris-Cl 17 pH 7.5, 50 mM KCl, 1 mM 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

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 / Number grids imaged: 1 / Number real images: 175 / Average exposure time: 15.0 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: 42343
CTF correctionSoftware - Name: CTFFIND (ver. 1.4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kx5


Details: .mrc file was generated in chimera. Model was low pass filtered to 50 angstrom
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 11063
FSC plot (resolution estimation)

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