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- EMDB-1102: Three-dimensional structure and regulation of the DNA-dependent p... -

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Basic information

Entry
Database: EMDB / ID: EMD-1102
TitleThree-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).
Map dataThis is the cryoEM 3D reconstruction of DNA-PKcs kinase.
Sample
  • Sample: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
  • Protein or peptide: DNA-dependent protein kinase catalytic subunit
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsRivera-Calzada A / Maman JP / Spagnolo L / Pearl LH / Llorca O
CitationJournal: Structure / Year: 2005
Title: Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).
Authors: Angel Rivera-Calzada / Joseph D Maman / Laura Spagnolo / Laurence H Pearl / Oscar Llorca /
Abstract: DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have ...DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have now generated an approximately 13 A three-dimensional map of DNA-PKcs, revealing the overall architecture and topology of the 4128 residue polypeptide chain and allowing location of domains. The highly conserved C-terminal PIKK catalytic domain forms a central structure from which FAT and FATC domains protrude. Conformational changes observed in these domains on DNA binding suggest that they transduce DNA-induced conformational changes to the catalytic core and regulate kinase activity. The N-terminal segments form long curved tubular-shaped domains based on helical repeats to create interacting surfaces required for macromolecular assembly. Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family.
History
DepositionDec 15, 2004-
Header (metadata) releaseDec 15, 2004-
Map releaseDec 15, 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: 2.829065583
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.829065583
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1102.gif

emd_1102.tif

Downloads & links

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Map

FileDownload / File: emd_1102.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the cryoEM 3D reconstruction of DNA-PKcs kinase.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 100 pix.
= 280. Å		2.8 Å/pix.
x 100 pix.
= 280. Å		2.8 Å/pix.
x 100 pix.
= 280. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.99 / Movie #1: 2.8290656
Minimum - Maximum0.0 - 8.253209999999999
Average (Standard dev.)0.127009 (±0.671219)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin111
Dimensions100100100
Spacing100100100
CellA=B=C: 280 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z280.000280.000280.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS111
NC/NR/NS100100100
D min/max/mean0.0008.2530.127

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Supplemental data

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Sample components

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Entire : Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs

EntireName: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
Components
  • Sample: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
  • Protein or peptide: DNA-dependent protein kinase catalytic subunit

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Supramolecule #1000: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs

SupramoleculeName: Human DNA-dependent protein kinase catalytic subunit, DNA-PKcs
type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightTheoretical: 470 KDa / Method: From its sequence

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Macromolecule #1: DNA-dependent protein kinase catalytic subunit

MacromoleculeName: DNA-dependent protein kinase catalytic subunit / type: protein_or_peptide / ID: 1 / Name.synonym: DNA-PKcs / 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: Nucleus
Molecular weightExperimental: 470 KDa
Recombinant expressionOrganism: HeLa

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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

Concentration0.2 mg/mL
BufferpH: 7.5 / Details: 50mM Tris-HCl, 50mM KCl, 1mM MgCl2.
GridDetails: 400 mesh carbon coated Rhodium-Copper grids
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home made plunger / Method: Blot for 1 second before plunging

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 90 K / Max: 90 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: objective astigmatism was corrected using the stigmator, CCD camera collected images and the Digital Micrograph software
DetailsMicroscope model: TECNAI G2 200 kV, FEI.
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 43 / Details: Images were averaged to 14 microns / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: side entry liquid-nitrogen cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: reverse phases for each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: EMAN
Details: CTF Corrected particles were subjected to 3D refinement as implemented in EMAN. The starting volume was generated by image classification of the whole data set into a few average images to ...Details: CTF Corrected particles were subjected to 3D refinement as implemented in EMAN. The starting volume was generated by image classification of the whole data set into a few average images to build a reconstruction using common lines.
Number images used: 7000
Final angle assignmentDetails: EMAN software criteria

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Atomic model buiding 1

Initial modelPDB ID:

1e8x

SoftwareName: SITUS
DetailsProtocol: Rigid Body. The domains were fitted using the COLORES command from SITUS
RefinementProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coeficient

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