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- PDB-7tyr: Cryo-EM structure of the basal state of the Artemis:DNA-PKcs comp... -

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Basic information

Entry
Database: PDB / ID: 7tyr
TitleCryo-EM structure of the basal state of the Artemis:DNA-PKcs complex (see COMPND 13/14)
Components
  • DNA-dependent protein kinase catalytic subunit
  • Protein artemis
KeywordsDNA BINDING PROTEIN / Kinase / nuclease
Function / homology
Function and homology information


positive regulation of platelet formation / single-stranded DNA endodeoxyribonuclease activity / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex ...positive regulation of platelet formation / single-stranded DNA endodeoxyribonuclease activity / T cell receptor V(D)J recombination / pro-B cell differentiation / small-subunit processome assembly / positive regulation of lymphocyte differentiation / DNA-dependent protein kinase complex / immature B cell differentiation / DNA-dependent protein kinase-DNA ligase 4 complex / nonhomologous end joining complex / immunoglobulin V(D)J recombination / regulation of smooth muscle cell proliferation / V(D)J recombination / double-strand break repair via alternative nonhomologous end joining / telomere capping / regulation of epithelial cell proliferation / Cytosolic sensors of pathogen-associated DNA / IRF3-mediated induction of type I IFN / regulation of hematopoietic stem cell differentiation / 5'-3' exonuclease activity / 5'-3' DNA exonuclease activity / U3 snoRNA binding / T cell lineage commitment / maturation of 5.8S rRNA / response to ionizing radiation / positive regulation of double-strand break repair via nonhomologous end joining / B cell lineage commitment / negative regulation of cGAS/STING signaling pathway / negative regulation of protein phosphorylation / ectopic germ cell programmed cell death / somitogenesis / interstrand cross-link repair / mitotic G1 DNA damage checkpoint signaling / peptidyl-threonine phosphorylation / telomere maintenance / activation of innate immune response / B cell differentiation / positive regulation of erythrocyte differentiation / positive regulation of translation / response to gamma radiation / Nonhomologous End-Joining (NHEJ) / small-subunit processome / regulation of circadian rhythm / protein destabilization / brain development / protein-DNA complex / protein modification process / double-strand break repair via nonhomologous end joining / cellular response to insulin stimulus / intrinsic apoptotic signaling pathway in response to DNA damage / rhythmic process / double-strand break repair / E3 ubiquitin ligases ubiquitinate target proteins / peptidyl-serine phosphorylation / T cell differentiation in thymus / heart development / double-stranded DNA binding / endonuclease activity / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / adaptive immune response / damaged DNA binding / Hydrolases; Acting on ester bonds / eukaryotic translation initiation factor 2alpha kinase activity / chromosome, telomeric region / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / non-specific serine/threonine protein kinase / protein kinase activity / protein phosphorylation / positive regulation of apoptotic process / protein domain specific binding / innate immune response / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / negative regulation of apoptotic process / chromatin / nucleolus / enzyme binding / Golgi apparatus / positive regulation of transcription by RNA polymerase II / protein-containing complex
Similarity search - Function
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 ...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 / : / FATC domain / PIK-related kinase, FAT / FAT domain / DNA repair metallo-beta-lactamase / DNA repair metallo-beta-lactamase / FATC / 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- 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 / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / Armadillo-like helical / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
DNA-dependent protein kinase catalytic subunit / Protein artemis
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsWatanabe, G. / Lieber, M.R. / Williams, D.R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA100504 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM118009 United States
CitationJournal: Nucleic Acids Res / Year: 2022
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
DepositionFeb 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.0Nov 13, 2024Group: Atomic model / Data collection ...Atomic model / Data collection / Refinement description / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / pdbx_poly_seq_scheme / refine / refine_hist
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.occupancy / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_poly_seq_scheme.auth_mon_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA-dependent protein kinase catalytic subunit
C: Protein artemis


Theoretical massNumber of molelcules
Total (without water)550,1672
Polymers550,1672
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA-dependent protein kinase catalytic subunit / DNA-PKcs / DNPK1 / p460


Mass: 469673.219 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HeLa-S3
References: UniProt: P78527, non-specific serine/threonine protein kinase
#2: Protein Protein artemis / DNA cross-link repair 1C protein / Protein A-SCID / SNM1 homolog C / hSNM1C / SNM1-like 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.) Homo sapiens (human) / Gene: DCLRE1C, ARTEMIS, ASCID, SCIDA, SNM1C / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q96SD1, Hydrolases; Acting on ester bonds
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: A complex of Artemis:DNA-PKcs / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMtris(hydroxymethyl)aminomethaneTris-HCl1
2100 mMSodium chlorideNaCl1
32 mM1,4-DithiothreitolDTT1
45.5 nMLauryl maltose neopentyl glycolLMNG1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.8 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
2DigitalMicrograph3.32.2403.0image acquisitionLatitude-S
7Coot0.9.6model fitting
9PHENIXdev-4383-000model refinement
13cryoSPARC3.2.0+2105113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103485 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 5LUQ

5luq


Pdb chain-ID: A / Accession code: 5LUQ / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.33 Å
Refinement stepCycle: LAST / Highest resolution: 3.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms31599 0 0 0 31599

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