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- PDB-8a2q: Structure of the DNA-bound FANCD2-FANCI complex containing phosph... -

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Basic information

Entry
Database: PDB / ID: 8a2q
TitleStructure of the DNA-bound FANCD2-FANCI complex containing phosphomimetic FANCI
Components
  • (DNA (29-MER)) x 2
  • FANCD2
  • Fanconi anemia complementation group I
KeywordsDNA BINDING PROTEIN / nucleic acid protein complex / DNA clamp / solenoid domains / Fanconi anemia / DNA repair / DNA damage / DNA inter strand crosslink / ubiquitination / ATR / pathway activation
Function / homology
Function and homology information


Fanconi Anemia Pathway in DNA repair / Fanconi Anemia Pathway / homologous chromosome pairing at meiosis / double-strand break repair involved in meiotic recombination / mitotic intra-S DNA damage checkpoint signaling / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / DNA repair / nucleoplasm / nucleus
Similarity search - Function
Fanconi anemia group I protein / FANCI solenoid 1 cap / FANCI solenoid 1 domain / FANCI helical domain 1 / FANCI helical domain 2 / FANCI solenoid 3 domain / FANCI solenoid 4 domain / FANCI solenoid 2 domain / FANCI solenoid 1 cap / FANCI solenoid 1 ...Fanconi anemia group I protein / FANCI solenoid 1 cap / FANCI solenoid 1 domain / FANCI helical domain 1 / FANCI helical domain 2 / FANCI solenoid 3 domain / FANCI solenoid 4 domain / FANCI solenoid 2 domain / FANCI solenoid 1 cap / FANCI solenoid 1 / FANCI solenoid 2 / FANCI solenoid 3 / FANCI solenoid 4 / FANCI helical domain 1 / FANCI helical domain 2 / Fanconi anaemia protein FANCD2 / Fanconi anaemia protein FancD2 nuclease
Similarity search - Domain/homology
DNA / DNA (> 10) / Fanconi anemia complementation group I / Fanconi anemia complementation group D2
Similarity search - Component
Biological speciesGallus gallus (chicken)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsPassmore, L.A. / Sijacki, T. / Alcon, P.
Funding support United Kingdom, Germany, European Union, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105192715 United Kingdom
German Research Foundation (DFG)329673113 Germany
European Molecular Biology Organization (EMBO)ALTF 692 2018European Union
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination.
Authors: Tamara Sijacki / Pablo Alcón / Zhuo A Chen / Stephen H McLaughlin / Shabih Shakeel / Juri Rappsilber / Lori A Passmore /
Abstract: DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which ...DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.
History
DepositionJun 6, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_entity_assembly / em_entity_assembly_molwt
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_entity_assembly.entity_id_list / _em_entity_assembly.source / _em_entity_assembly_molwt.entity_assembly_id
Revision 1.2Oct 5, 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: FANCD2
B: Fanconi anemia complementation group I
S: DNA (29-MER)
T: DNA (29-MER)


Theoretical massNumber of molelcules
Total (without water)342,1854
Polymers342,1854
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein FANCD2 /


Mass: 164731.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: FANCD2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F1NP22
#2: Protein Fanconi anemia complementation group I


Mass: 150357.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: FANCI / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B0I564
#3: DNA chain DNA (29-MER)


Mass: 13554.697 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (29-MER)


Mass: 13541.715 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D, S561D and T567D)COMPLEXall0MULTIPLE SOURCES
2FANCD2COMPLEX#11RECOMBINANTThe consensus complex map of DNA-bound FANCD2-FANCI3D was subjected to signal subtraction of DNA and FANCI and then refined to obtain a higher resolution map of FANCD2.
3FANCI3DCOMPLEX#2-#41MULTIPLE SOURCESFANCI containing phosphomimetic mutations ((S558D, S561D and T567D). The consensus complex map of DNA-bound FANCD2-FANCI3D was subjected to signal subtraction of DNA and FANCD2 and then refined to obtain a higher resolution map of FANCI3D.
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.341 MDaNO
220.164 MDaNO
330.150 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Gallus gallus (chicken)9031
32Gallus gallus (chicken)9031
43Gallus gallus (chicken)9031
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
250 mMHEPESC8H18N2O4S1
31 mMTCEPC9H15O6P1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14842
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.6particle selection
4CTFFIND4.1CTF correction
7Coot0.9model fitting
8PHENIX1.19model fitting
9UCSF Chimera1.15.0model fitting
10UCSF ChimeraX1.1.1model fitting
11ISOLDEmodel fitting
13PHENIX1.19model refinement
14RELION3.1initial Euler assignment
15RELION3.1final Euler assignment
16RELION3.1classification
17RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1356378
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 197436 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: For dsDNA (chain S and T)an ideal B form DNA was generated and fitted into the density using the same softwares used to fit FANCD2 and FANCI chains (chain A and B) into the corresponding density.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16TNF

6tnf

A1
26TNF

6tnf

B1

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