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- PDB-7tyr: Cryo-EM structure of the basal state of the Artemis:DNA-PKcs comp... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7tyr | |||||||||
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Title | Cryo-EM structure of the basal state of the Artemis:DNA-PKcs complex (see COMPND 13/14) | |||||||||
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![]() | DNA BINDING PROTEIN / Kinase / nuclease | |||||||||
Function / homology | ![]() 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 / single-stranded DNA endodeoxyribonuclease activity / immunoglobulin V(D)J recombination / nonhomologous end joining complex / regulation of smooth muscle cell proliferation / V(D)J recombination / Cytosolic sensors of pathogen-associated DNA / regulation of epithelial cell proliferation / IRF3-mediated induction of type I IFN / telomere capping / 5'-3' exonuclease activity / regulation of hematopoietic stem cell differentiation / U3 snoRNA binding / 5'-3' DNA exonuclease activity / T cell lineage commitment / negative regulation of cGAS/STING signaling pathway / response to ionizing radiation / double-strand break repair via alternative nonhomologous end joining / maturation of 5.8S rRNA / B cell lineage commitment / positive regulation of double-strand break repair via nonhomologous end joining / ectopic germ cell programmed cell death / somitogenesis / interstrand cross-link repair / mitotic G1 DNA damage checkpoint signaling / activation of innate immune response / telomere maintenance / B cell differentiation / positive regulation of erythrocyte differentiation / negative regulation of protein phosphorylation / small-subunit processome / positive regulation of translation / protein-DNA complex / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / peptidyl-threonine phosphorylation / protein destabilization / protein modification process / brain development / regulation of circadian rhythm / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / intrinsic apoptotic signaling pathway in response to DNA damage / double-strand break repair / rhythmic process / E3 ubiquitin ligases ubiquitinate target proteins / heart development / T cell differentiation in thymus / double-stranded DNA binding / peptidyl-serine phosphorylation / endonuclease activity / adaptive immune response / RNA polymerase II-specific DNA-binding transcription factor binding / transcription regulator complex / damaged DNA binding / chromosome, telomeric region / Hydrolases; Acting on ester bonds / 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 / Golgi apparatus / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / RNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å | |||||||||
![]() | Watanabe, G. / Lieber, M.R. / Williams, D.R. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural analysis of the basal state of the Artemis:DNA-PKcs complex. Authors: Go Watanabe / Michael R Lieber / Dewight R Williams / ![]() Abstract: Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. ...Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 971.9 KB | Display | ![]() |
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PDB format | ![]() | 774.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 26192MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 469673.219 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() References: UniProt: P78527, non-specific serine/threonine protein kinase |
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#2: Protein | Mass: 80493.352 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Please use a 'blurred' map of the EMD-26192 entry to see the density of the Artemis catalytic region Source: (gene. exp.) ![]() ![]() References: UniProt: Q96SD1, Hydrolases; Acting on ester bonds |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: A complex of Artemis:DNA-PKcs / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse. | |||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Humidity: 73 % / Chamber temperature: 295 K Details: Plunge-freeze was performed using a home-made manual plunger at typical indoor humidity (Los Angeles, CA) and at room temperature. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 46296 X / Nominal defocus max: 3000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3.8 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||
3D reconstruction | Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103485 / Symmetry type: POINT | |||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | |||||||||||||||||||||||||
Atomic model building | PDB-ID: 5LUQ Pdb chain-ID: A |