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- EMDB-1209: Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-1209
TitleThree-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair.
Map data3D reconstruction of the DNA-bound DNAPKcs-Ku70-Ku80 complex obtained from negatively stained samples
Sample
  • Sample: DNA-bound DNAPKcs-Ku70-Ku80 complex
  • Protein or peptide: DNA-PKcs
  • Protein or peptide: Ku70
  • Protein or peptide: Ku80
  • DNA: DNA
Function / homology: / Ku70 / Phosphatidylinositol 3-/4-kinase, catalytic domain / double-strand break repair via nonhomologous end joining / double-strand break repair / protein kinase activity
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsSpagnolo L / Rivera-Calzada A / Pearl LH / Llorca O
CitationJournal: Mol Cell / Year: 2006
Title: Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair.
Authors: Laura Spagnolo / Angel Rivera-Calzada / Laurence H Pearl / Oscar Llorca /
Abstract: DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by ...DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.
History
DepositionMar 30, 2006-
Header (metadata) releaseMar 31, 2006-
Map releaseMar 31, 2008-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.439739195
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.439739195
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1209.gif

Downloads & links

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Map

FileDownload / File: emd_1209.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the DNA-bound DNAPKcs-Ku70-Ku80 complex obtained from negatively stained samples
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.4 Å/pix.
x 72 pix.
= 312.4 Å		4.4 Å/pix.
x 72 pix.
= 312.4 Å		4.4 Å/pix.
x 72 pix.
= 312.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.4 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.5 / Movie #1: 3.4397392
Minimum - Maximum-8.42113 - 13.4579
Average (Standard dev.)0.000147287 (±0.999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 312.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z717171
origin x/y/z0.0000.0000.000
length x/y/z312.400312.400312.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-96-96-96
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-8.42113.4580.000

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Supplemental data

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Sample components

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Entire : DNA-bound DNAPKcs-Ku70-Ku80 complex

EntireName: DNA-bound DNAPKcs-Ku70-Ku80 complex
Components
  • Sample: DNA-bound DNAPKcs-Ku70-Ku80 complex
  • Protein or peptide: DNA-PKcs
  • Protein or peptide: Ku70
  • Protein or peptide: Ku80
  • DNA: DNA

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Supramolecule #1000: DNA-bound DNAPKcs-Ku70-Ku80 complex

SupramoleculeName: DNA-bound DNAPKcs-Ku70-Ku80 complex / type: sample / ID: 1000
Oligomeric state: One DNA-PKcs molecule binds one Ku70 and one Ku80 on one DNA molecule
Number unique components: 4
Molecular weightTheoretical: 650 KDa

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Macromolecule #1: DNA-PKcs

MacromoleculeName: DNA-PKcs / type: protein_or_peptide / ID: 1
Name.synonym: DNA-dependent Protein Kinase catalytic subunit
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus / Location in cell: Nuclear
Molecular weightExperimental: 470 KDa
Recombinant expressionOrganism: HeLa cells
SequenceGO: protein kinase activity
InterPro: Phosphatidylinositol 3-/4-kinase, catalytic domain

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Macromolecule #2: Ku70

MacromoleculeName: Ku70 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus / Location in cell: Nuclear
Molecular weightExperimental: 70 KDa
Recombinant expressionOrganism: HeLa cells
SequenceGO: double-strand break repair via nonhomologous end joining
InterPro: Ku70

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Macromolecule #3: Ku80

MacromoleculeName: Ku80 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus / Location in cell: Nuclear
Molecular weightExperimental: 80 KDa
Recombinant expressionOrganism: HeLa cells
SequenceGO: double-strand break repair / InterPro: INTERPRO: IPR011210

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Macromolecule #4: DNA

MacromoleculeName: DNA / type: dna / ID: 4 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
SequenceString:
ACGCGTGCGG CCATAATAAT AGTTTTTAGT TTATTGGGCG CG

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 0.5 mM DTT, 0.25 mM EDTA, 0.0005% beta-octylglucoside, 25% glycerol, 100 mM NaCl
StainingType: NEGATIVE
Details: The sample was applied to carbon-coated grids after glow-discharge and negatively stained with 1% uranyl acetate
GridDetails: 400 mesh Rhodium-copper grids
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeJEOL 1230
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.9 mm / Nominal magnification: 50000
Sample stageSpecimen holder: JEOL type M / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: correction with FFT and CCD camera
DetailsMicroscope: JEOL 1230 operated at 100 Kv Specimen holder, JEOL type M: 207EM-11020.
Image recordingCategory: FILM / Film or detector model: KODAK 4489 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 84 / Details: Scanner: MINOLTA Dimage Scan Multi Pro scanner / Bits/pixel: 16
Tilt angle min0
Tilt angle max0

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 14239

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