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- EMDB-4425: Cryo-EM structure of DNA-PKcs -

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Basic information

Entry
Database: EMDB / ID: EMD-4425
TitleCryo-EM structure of DNA-PKcs
Map data
SampleDNA-dependent protein kinase catalytic subunit
SourceHomo (humans)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsWu Q / Blundell TL
CitationJournal: Prog. Biophys. Mol. Biol. / Year: 2019
Title: Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways.
Authors: Qian Wu / Shikang Liang / Takashi Ochi / Dimitri Y Chirgadze / Juha T Huiskonen / Tom L Blundell /
Abstract: DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End ...DNA double-strand breaks (DSBs), generated by ionizing radiation, reactive oxygen species and DNA replication across nicks, are the most severe DNA damage in eukaryotic cells. Non-Homologous End Joining repairs DNA double-strand breaks directly without a template and so can take place at any point in the cell cycle. Ku70/80 heterodimers rapidly assemble around broken DNA ends, allowing DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to be recruited and facilitating synapsis of broken DNA ends. This then provides a stage for end-processing and ligation. Here we review progress leading in 2017 to the medium resolution X-ray structure of DNA-PKcs, a single polypeptide chain of 4128 amino acids. This was followed quickly by chain tracing of cryo-EM structures of DNA-PKcs in complex with Ku and DNA. We discuss how combination of structural information from X-ray and cryo-EM studies can produce a working model for complex multicomponent molecular assemblies such as those found in DNA-double-strand-break repair.
DateDeposition: Nov 16, 2018 / Header (metadata) release: May 8, 2019 / Map release: May 8, 2019 / Update: May 8, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0906
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0906
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4425.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 220 pix.
= 314.6 Å
1.43 Å/pix.
x 220 pix.
= 314.6 Å
1.43 Å/pix.
x 220 pix.
= 314.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.0906 / Movie #1: 0.0906
Minimum - Maximum-0.23143104 - 0.48773596
Average (Standard dev.)0.0020121848 (±0.017275512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 314.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z314.600314.600314.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2310.4880.002

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Supplemental data

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Sample components

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Entire DNA-dependent protein kinase catalytic subunit

EntireName: DNA-dependent protein kinase catalytic subunit / Number of components: 2

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Component #1: cellular-component, DNA-dependent protein kinase catalytic subunit

Cellular-componentName: DNA-dependent protein kinase catalytic subunit / Recombinant expression: No
SourceSpecies: Homo (humans)

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Component #2: protein, DNA-dependent protein kinase catalytic subunit

ProteinName: DNA-dependent protein kinase catalytic subunit / Recombinant expression: No
SourceSpecies: Homo (humans)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/mL
Buffer solution: 20 mM Hepes pH 7.6, 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, 5 mM DTT
pH: 7.6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 129117
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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