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- EMDB-1271: Structural model of full-length human Ku70-Ku80 heterodimer and i... -

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Basic information

Entry
Database: EMDB / ID: EMD-1271
TitleStructural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs.
Map dataFrontal view of the Ku+DNA volume at a thresold of 2.5
Sample
  • Sample: Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bound to DNA
  • Protein or peptide: Ku70
  • Protein or peptide: Ku80
  • DNA: DNA
Function / homology: / Ku70/Ku80 beta-barrel domain / nucleus
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsRivera-Calzada A / Spagnolo L / Pearl LH / Llorca O
CitationJournal: EMBO Rep / Year: 2007
Title: Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs.
Authors: Angel Rivera-Calzada / Laura Spagnolo / Laurence H Pearl / Oscar Llorca /
Abstract: Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein ...Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.
History
DepositionOct 3, 2006-
Header (metadata) releaseOct 3, 2006-
Map releaseOct 3, 2006-
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: 2.371905906
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.371905906
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1271.gif

Downloads & links

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Map

FileDownload / File: emd_1271.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFrontal view of the Ku+DNA volume at a thresold of 2.5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 96 pix.
= 203.52 Å		2.12 Å/pix.
x 96 pix.
= 203.52 Å		2.12 Å/pix.
x 96 pix.
= 203.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.14 / Movie #1: 2.3719059
Minimum - Maximum-2.33323 - 13.3996
Average (Standard dev.)0.206044 (±1.0865)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 203.52 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z203.520203.520203.520
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-2.33313.4000.206

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Supplemental data

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Sample components

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Entire : Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bo...

EntireName: Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bound to DNA
Components
  • Sample: Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bound to DNA
  • Protein or peptide: Ku70
  • Protein or peptide: Ku80
  • DNA: DNA

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Supramolecule #1000: Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bo...

SupramoleculeName: Ku70-Ku80 heterodimer purified from HeLa cell nuclear extracts bound to DNA
type: sample / ID: 1000
Oligomeric state: Heterodimer of Ku70 and Ku80 complexed bound to DNA
Number unique components: 3
Molecular weightExperimental: 152 KDa

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Macromolecule #1: Ku70

MacromoleculeName: Ku70 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 70 KDa
SequenceGO: GO: 0005624 / InterPro: Ku70/Ku80 beta-barrel domain

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Macromolecule #2: Ku80

MacromoleculeName: Ku80 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus
Molecular weightExperimental: 82 KDa
SequenceGO: nucleus / InterPro: Ku70/Ku80 beta-barrel domain

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Details: 54 bp blunt-ended dsDNA 5' biotinilated / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
SequenceString:
GGCCGCACGC GTCCACCATG GGGTACAACT ACGATCTAGC TTCATGCACC GGAC

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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

Concentration0.068 mg/mL
BufferDetails: 50 mM Tris pH 7.5, 10% glycerol, 1mM DTT, 5 mM EDTA, 0.5mM Mg2Cl, 100mM NaCl, 20mM KCl
StainingType: NEGATIVE
Details: A few microliters of the DNA-bound Ku complexes were adsorbed to glow discharged carbon coated grids and negatively stained using 1% uranyl acetate.
GridDetails: 400 mesh Copper/Palladium grid
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeJEOL 1230
Alignment procedureLegacy - Astigmatism: correction with FFT and CCD camera
DetailsMicroscope used: JEOL1230
DateApr 20, 2005
Image recordingCategory: FILM / Film or detector model: KODAK 4489 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Details: Scanner: MINOLTA Dimage Scan Multi Pro scanner / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER / Tilt angle max: 35

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Image processing

DetailsPurified Ku and a ~10- fold molar excess of DNA were incubated at room temperature for 20 minutes.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 8285

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Atomic model buiding 1

Initial modelPDB ID:

1jey

SoftwareName: Situs
DetailsProtocol: Rigid Body. Fitting performed using Situs
RefinementProtocol: RIGID BODY FIT / Target criteria: R-factor

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