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- EMDB-5831: CryoEM structure of DNA-PKcs -

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Basic information

Entry
Database: EMDB / ID: EMD-5831
TitleCryoEM structure of DNA-PKcs
Map dataReconstruction of DNA-PKcs
Sample
  • Sample: DNA-PKcs
  • Protein or peptide: DNA-Dependent Protein Kinase Catalytic Subunit
KeywordsCryo-electron microscopy / cryoEM / single particle reconstruction / flexibility / heterogeneity / eigenimage sorting / protein/DNA complexes
Function / homology
Function and homology information


MHC class II antigen presentation / Neutrophil degranulation / entry into host cell by a symbiont-containing vacuole / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / protein autoprocessing / transport vesicle / protein processing / double-strand break repair via nonhomologous end joining / aspartic-type endopeptidase activity / proteolysis
Similarity search - Function
DNA-dependent protein kinase catalytic subunit, CC3 / Pepsin-like domain / PIK-related kinase, FAT / FATC domain / PIK-related kinase / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3-/4-kinase, catalytic domain / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain ...DNA-dependent protein kinase catalytic subunit, CC3 / Pepsin-like domain / PIK-related kinase, FAT / FATC domain / PIK-related kinase / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3-/4-kinase, catalytic domain / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. / Armadillo-like helical / Armadillo-type fold / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsVillarreal SA / Stewart PL
CitationJournal: J Struct Biol / Year: 2014
Title: CryoEM and image sorting for flexible protein/DNA complexes.
Authors: Seth A Villarreal / Phoebe L Stewart /
Abstract: Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder ...Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.
History
DepositionDec 10, 2013-
Header (metadata) releaseJan 1, 2014-
Map releaseJan 1, 2014-
UpdateJul 16, 2014-
Current statusJul 16, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.438
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.438
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5831.png

Downloads & links

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Map

FileDownload / File: emd_5831.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of DNA-PKcs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 300 pix.
= 531. Å		1.77 Å/pix.
x 300 pix.
= 531. Å		1.77 Å/pix.
x 300 pix.
= 531. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.438 / Movie #1: 0.438
Minimum - Maximum-1.02578521 - 1.49648023
Average (Standard dev.)0.02785548 (±0.0465954)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-149-149-149
Dimensions300300300
Spacing300300300
CellA=B=C: 531.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z531.000531.000531.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-149-149-149
NC/NR/NS300300300
D min/max/mean-1.0261.4960.028

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Supplemental data

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Sample components

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Entire : DNA-PKcs

EntireName: DNA-PKcs
Components
  • Sample: DNA-PKcs
  • Protein or peptide: DNA-Dependent Protein Kinase Catalytic Subunit

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Supramolecule #1000: DNA-PKcs

SupramoleculeName: DNA-PKcs / type: sample / ID: 1000 / Details: The sample was monodisperse. / Number unique components: 1
Molecular weightTheoretical: 469 KDa

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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: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus
Molecular weightTheoretical: 469 KDa
SequenceUniProtKB: Plasmepsin X
GO: double-strand break repair via nonhomologous end joining
InterPro: Armadillo-like helical, Armadillo-type fold, FATC domain, Protein kinase-like domain superfamily, DNA-dependent protein kinase catalytic subunit, CC3, Phosphatidylinositol 3-/4-kinase, ...InterPro: Armadillo-like helical, Armadillo-type fold, FATC domain, Protein kinase-like domain superfamily, DNA-dependent protein kinase catalytic subunit, CC3, Phosphatidylinositol 3-/4-kinase, catalytic domain, Phosphatidylinositol 3/4-kinase, conserved site, PIK-related kinase, FAT, PIK-related kinase

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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

VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
DateDec 31, 2006
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 254669 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: -6.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 200000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected with an in-house script and processed using Frealign and 3D Fourier Space v2.
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.6 Å / Resolution method: OTHER / Software - Name: Frealign, Imagic / Number images used: 153545

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