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- EMDB-21982: PARP2/HPF1_Nucleosome complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21982
TitlePARP2/HPF1_Nucleosome complex
Map dataSubstrate class of PARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Sample
  • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
    • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1
    • Complex: DNA
    • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2
Biological speciesAfrican clawed frog (African clawed frog) / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsHalic M / Bilokapic S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM135599-01 United States
CitationJournal: Nature / Year: 2020
Title: Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin.
Authors: Silvija Bilokapic / Marcin J Suskiewicz / Ivan Ahel / Mario Halic /
Abstract: Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR). In the DNA damage responses, ...Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR). In the DNA damage responses, this post-translational modification occurs predominantly on serine residues and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments.
History
DepositionMay 16, 2020-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateOct 7, 2020-
Current statusOct 7, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21982.png

Downloads & links

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Map

FileDownload / File: emd_21982.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubstrate class of PARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy EMDB: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.10733052 - 0.35010883
Average (Standard dev.)0.00005725578 (±0.003706681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z508.800508.800508.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1070.3500.000

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Supplemental data

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Sample components

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Entire : Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Hi...

EntireName: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
Components
  • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
    • Complex: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1
    • Complex: DNA
    • Complex: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2

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Supramolecule #1: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Hi...

SupramoleculeName: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1, Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2/DNA Complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8 / Details: PARP2/HPF1 from PARP2/HPF1_Nucleosome complex
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1

SupramoleculeName: Histone H3.2, Histone H4, Histone H2A type 1, Histone H2B 1.1
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: African clawed frog (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: synthetic construct (others)

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Supramolecule #4: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2

SupramoleculeName: Histone PARylation factor 1, Poly [ADP-ribose] polymerase 2
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #7-#8
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 934000
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11000

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