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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: 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
SampleKu70-Ku80 heterodimer purified from HeLa cell nuclear extracts bound to DNA:
Ku70 / Ku80 / nucleic-acidNucleic acid
Function / homologygo:0005624: / Ku70/Ku80 beta-barrel domain / nucleus
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / 25 Å resolution
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
DateDeposition: Oct 3, 2006 / Header (metadata) release: Oct 3, 2006 / Map release: Oct 3, 2006 / Last update: May 26, 2011

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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

Downloads & links

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Map

Fileemd_1271.map.gz (map file in CCP4 format, 3457 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
96 pix
2.12 Å/pix.
= 203.52 Å
96 pix
2.12 Å/pix.
= 203.52 Å
96 pix
2.12 Å/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
Contour Level:1.14, 2.3719059 (movie #1):
Minimum - Maximum-2.33323 - 13.3996
Average (Standard dev.)0.206044 (1.0865)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions969696
Origin-48-48-48
Limit474747
Spacing969696
CellA=B=C: 203.52 Å
α=β=γ: 90 deg.

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
Oligomeric State: Heterodimer of Ku70 and Ku80 complexed bound to DNA
Number of components: 3
MassExperimental: 152 kDa

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Component #1: protein, Ku70

ProteinName: Ku70 / Number of Copies: 1 / Recombinant expression: No
MassExperimental: 70 kDa
SourceSpecies: Homo sapiens (human)
Source (natural)Organelle: Nucleus / Cell: HeLa
External referencesGene Ontology: GO: 0005624 / InterPro: Ku70/Ku80 beta-barrel domain

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Component #2: protein, Ku80

ProteinName: Ku80 / Recombinant expression: No / Number of Copies: 1
MassExperimental: 82 kDa
SourceSpecies: Homo sapiens (human)
Source (natural)Organelle: Nucleus / Cell: HeLa
External referencesInterPro: Ku70/Ku80 beta-barrel domain / Gene Ontology: nucleus

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Component #3: nucleic-acid, DNA

Nucleic-acidName: DNA / Class: DNA / Details: 54 bp blunt-ended dsDNA 5' biotinilated / Structure: DOUBLE HELIX
Sequence:
GGCCGCACGC GTCCACCATG GGGTACAACT ACGATCTAGC TTCATGCACC GGAC

Synthetic: Yes

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.068 mg/ml
Buffer solution: 50 mM Tris pH 7.5, 10% glycerol, 1mM DTT, 5 mM EDTA, 0.5mM Mg2Cl, 100mM NaCl, 20mM KCl
Support film400 mesh Copper/Palladium grid
StainingA few microliters of the DNA-bound Ku complexes were adsorbed to glow discharged carbon coated grids and negatively stained using 1% uranyl acetate.
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 1230 / Date: Apr 20, 2005 / Details: Microscope used: JEOL1230
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Astigmatism: correction with FFT and CCD camera / Cs: 2.9 mm / Imaging mode: BRIGHT FIELD
Specimen HolderHolder: Eucentric / Model: OTHER / Tilt Angle: 0 - 35 deg.
CameraDetector: KODAK 4489 FILM

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Image acquisition

Image acquisitionScanner: OTHER / Sampling size: 10 microns / Bit depth: 16 / Details: Scanner: MINOLTA Dimage Scan Multi Pro scanner

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 8285
Details: Purified Ku and a ~10- fold molar excess of DNA were incubated at room temperature for 20 minutes.
Applied symmetry: C1 (asymmetric)
3D reconstructionSoftware: EMAN / Resolution: 25 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Situs / Refinement protocol: rigid body / Target criteria: R-factor
Details: Protocol: Rigid Body. Fitting performed using Situs
Input PDB model: 1JEY

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