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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


MHC class II antigen presentation / nonhomologous end joining complex / single-stranded DNA endodeoxyribonuclease activity / Neutrophil degranulation / V(D)J recombination / entry into host cell by a symbiont-containing vacuole / 5'-3' exonuclease activity / 5'-3' DNA exonuclease activity / response to ionizing radiation / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases ...MHC class II antigen presentation / nonhomologous end joining complex / single-stranded DNA endodeoxyribonuclease activity / Neutrophil degranulation / V(D)J recombination / entry into host cell by a symbiont-containing vacuole / 5'-3' exonuclease activity / 5'-3' DNA exonuclease activity / response to ionizing radiation / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / protein autoprocessing / interstrand cross-link repair / transport vesicle / telomere maintenance / B cell differentiation / Nonhomologous End-Joining (NHEJ) / protein processing / double-strand break repair via nonhomologous end joining / endonuclease activity / adaptive immune response / damaged DNA binding / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / Golgi apparatus / proteolysis / nucleoplasm
Similarity search - Function
Pepsin-like domain / DNA repair metallo-beta-lactamase / DNA repair metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / Eukaryotic aspartyl protease / Aspartic peptidase A1 family / Peptidase family A1 domain / Peptidase family A1 domain profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily
Similarity search - Domain/homology
Plasmepsin X / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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

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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

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