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- EMDB-5832: CryoEM structure of DNA-PKcs/DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-5832
TitleCryoEM structure of DNA-PKcs/DNA
Map dataReconstruction of DNA-PKcs/DNA
Sample
  • Sample: DNA-PKcs/DNA
  • Protein or peptide: DNA-Dependent Protein Kinase Catalytic Subunit
  • DNA: DNA
KeywordsCryo-electron microscopy / cryoEM / single particle reconstruction / flexibility / heterogeneity / eigenimage sorting / protein/DNA complexes
Function / homology
Function and homology information


positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex ...positive regulation of platelet formation / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase activity / histone H2AXS139 kinase activity / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / immunoglobulin V(D)J recombination / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / double-strand break repair via alternative nonhomologous end joining / Cytosolic sensors of pathogen-associated DNA / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / U3 snoRNA binding / regulation of hematopoietic stem cell differentiation / maturation of 5.8S rRNA / T cell lineage commitment / negative regulation of cGAS/STING signaling pathway / B cell lineage commitment / positive regulation of double-strand break repair via nonhomologous end joining / ectopic germ cell programmed cell death / somitogenesis / mitotic G1 DNA damage checkpoint signaling / telomere maintenance / activation of innate immune response / small-subunit processome / positive regulation of translation / negative regulation of protein phosphorylation / positive regulation of erythrocyte differentiation / protein-DNA complex / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / brain development / peptidyl-threonine phosphorylation / protein destabilization / protein modification process / regulation of circadian rhythm / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / rhythmic process / double-strand break repair / intrinsic apoptotic signaling pathway in response to DNA damage / E3 ubiquitin ligases ubiquitinate target proteins / T cell differentiation in thymus / heart development / double-stranded DNA binding / peptidyl-serine phosphorylation / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / chromosome, telomeric region / non-specific serine/threonine protein kinase / protein kinase activity / positive regulation of apoptotic process / protein domain specific binding / protein phosphorylation / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / chromatin / nucleolus / negative regulation of apoptotic process / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / RNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol
Similarity search - Function
DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC1/2 ...DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-dependent protein kinase catalytic subunit, catalytic domain / DNA-dependent protein kinase catalytic subunit, CC5 / DNA-dependent protein kinase catalytic subunit, CC1/2 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC3 / DNA-PKcs, CC5 / DNA-PKcs, N-terminal / DNA-dependent protein kinase catalytic subunit, CC1/2 / NUC194 / PIK-related kinase, FAT / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain / PIK-related kinase / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Armadillo-like helical / Armadillo-type fold / Armadillo-type fold / Protein kinase-like domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
DNA-dependent protein kinase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 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: 0.531
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.531
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5832.png

Downloads & links

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Map

FileDownload / File: emd_5832.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of DNA-PKcs/DNA
Voxel sizeX=Y=Z: 2.35 Å
Density
Contour LevelBy AUTHOR: 0.531 / Movie #1: 0.531
Minimum - Maximum-1.73263061 - 1.84021735
Average (Standard dev.)0.03140974 (±0.14355543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-63-63-63
Dimensions128128128
Spacing128128128
CellA=B=C: 300.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.352.352.35
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z300.800300.800300.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-63-63-63
NC/NR/NS128128128
D min/max/mean-1.7331.8400.031

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Supplemental data

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Sample components

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Entire : DNA-PKcs/DNA

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

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Supramolecule #1000: DNA-PKcs/DNA

SupramoleculeName: DNA-PKcs/DNA / type: sample / ID: 1000 / Details: The sample was monodisperse. / Number unique components: 2
Molecular weightTheoretical: 534 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: DNA-dependent protein kinase catalytic subunit
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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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Details: biotinylated with streptavidin / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightTheoretical: 65 KDa
SequenceString:
AGGCTGTGTC CTCAGAGG

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 254669 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
DateDec 31, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: Frealign, Imagic / Number images used: 35163
DetailsThe particles were selected with an in-house script and processed using Frealign and 3D Fourier Space v2.

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