+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5833 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | CryoEM structure of DNA-PKcs/Ku/DNA | |||||||||
Map data | Reconstruction of DNA-PKcs/Ku/DNA | |||||||||
Sample |
| |||||||||
Keywords | Cryo-electron microscopy / cryoEM / single particle reconstruction / flexibility / heterogeneity / eigenimage sorting / protein/DNA complexes | |||||||||
Function / homology | Function and homology information Ku70:Ku80 complex / negative regulation of t-circle formation / positive regulation of platelet formation / DNA end binding / 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 ...Ku70:Ku80 complex / negative regulation of t-circle formation / positive regulation of platelet formation / DNA end binding / 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 / cellular response to X-ray / immunoglobulin V(D)J recombination / nonhomologous end joining complex / nuclear telomere cap 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 / recombinational repair / regulation of telomere maintenance / U3 snoRNA binding / regulation of hematopoietic stem cell differentiation / positive regulation of neurogenesis / protein localization to chromosome, telomeric region / cellular response to fatty acid / hematopoietic stem cell proliferation / cellular hyperosmotic salinity response / maturation of 5.8S rRNA / T cell lineage commitment / negative regulation of cGAS/STING signaling pathway / telomeric DNA binding / positive regulation of catalytic activity / B cell lineage commitment / 2-LTR circle formation / positive regulation of double-strand break repair via nonhomologous end joining / site of DNA damage / enzyme activator activity / hematopoietic stem cell differentiation / positive regulation of protein kinase activity / ectopic germ cell programmed cell death / regulation of DNA repair / ATP-dependent activity, acting on DNA / somitogenesis / positive regulation of telomerase activity / mitotic G1 DNA damage checkpoint signaling / positive regulation of telomere maintenance via telomerase / DNA helicase activity / activation of innate immune response / telomere maintenance / cellular response to leukemia inhibitory factor / neurogenesis / 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) / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / brain development / peptidyl-threonine phosphorylation / protein destabilization / protein modification process / cellular response to gamma radiation / 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 / secretory granule lumen / DNA recombination / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / damaged DNA binding / chromosome, telomeric region / transcription cis-regulatory region binding / non-specific serine/threonine protein kinase / protein kinase activity / response to xenobiotic stimulus / ribonucleoprotein complex / positive regulation of apoptotic process / protein domain specific binding / protein phosphorylation / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / negative regulation of DNA-templated transcription / DNA damage response / ubiquitin protein ligase binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 21.4 Å | |||||||||
Authors | Villarreal SA / Stewart PL | |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5833.map.gz | 50.4 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-5833-v30.xml emd-5833.xml | 13.7 KB 13.7 KB | Display Display | EMDB header |
Images | emd_5833.png | 96.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5833 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5833 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_5833.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of DNA-PKcs/Ku/DNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.262 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : DNA-PKcs/Ku/DNA
Entire | Name: DNA-PKcs/Ku/DNA |
---|---|
Components |
|
-Supramolecule #1000: DNA-PKcs/Ku/DNA
Supramolecule | Name: DNA-PKcs/Ku/DNA / type: sample / ID: 1000 / Details: The sample was monodisperse. / Number unique components: 4 |
---|---|
Molecular weight | Theoretical: 701 KDa |
-Macromolecule #1: DNA-Dependent Protein Kinase Catalytic Subunit
Macromolecule | Name: 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 weight | Theoretical: 469 KDa |
Sequence | UniProtKB: 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 |
-Macromolecule #3: Ku 70
Macromolecule | Name: Ku 70 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes / Database: NCBI |
---|---|
Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus |
Molecular weight | Theoretical: 70 KDa |
Sequence | UniProtKB: Ku70-binding protein / GO: regulation of DNA repair 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 |
-Macromolecule #4: Ku 86
Macromolecule | Name: Ku 86 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes / Database: NCBI |
---|---|
Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus |
Molecular weight | Theoretical: 86 KDa |
Sequence | UniProtKB: X-ray repair cross-complementing protein 5 / GO: regulation of DNA repair 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 |
-Macromolecule #2: DNA
Macromolecule | Name: DNA / type: dna / ID: 2 / Details: biotinylated with streptavidin / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
---|---|
Source (natural) | Organism: Homo sapiens (human) / synonym: human |
Molecular weight | Theoretical: 76 KDa |
Sequence | String: AGGCTGTGTT AAGTATCTGC GCTCGCCCTC AGAGG |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Vitrification | Cryogen name: ETHANE / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER |
---|
-Electron microscopy
Microscope | FEI POLARA 300 |
---|---|
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 198939 / 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: 155000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Temperature | Min: 80 K / Max: 105 K / Average: 100 K |
Date | Dec 31, 2010 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Bits/pixel: 16 |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each micrograph |
---|---|
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.4 Å / Resolution method: OTHER / Software - Name: Frealign, Imagic / Number images used: 19825 |
Details | The particles were selected with an in-house script and processed using Frealign and 3D Fourier Space v2. |