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- EMDB-15101: Structure of the DNA-bound FANCD2-FANCI complex containing phosph... -

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Basic information

Entry
Database: EMDB / ID: EMD-15101
TitleStructure of the DNA-bound FANCD2-FANCI complex containing phosphomimetic FANCI
Map data
Sample
  • Complex: DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D, S561D and T567D)
    • Complex: FANCD2
      • Protein or peptide: FANCD2
    • Complex: FANCI3D
      • Protein or peptide: Fanconi anemia complementation group I
      • DNA: DNA (29-MER)
      • DNA: DNA (29-MER)
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
Fanconi anemia complementation group I / Fanconi anemia complementation group D2
Similarity search - Component
Biological speciesGallus gallus (chicken) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsPassmore LA / Sijacki T / Alcon P
Funding support United Kingdom, Germany, European Union, 3 items
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, 2022-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15101.png

Downloads & links

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Map

FileDownload / File: emd_15101.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å		0.86 Å/pix.
x 300 pix.
= 258. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
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Contour LevelBy AUTHOR: 0.0056
Minimum - Maximum-0.013601029 - 0.043098476
Average (Standard dev.)0.00018214856 (±0.0015498232)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 258.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15101_msk_1.map
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Additional map: #4

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Additional map: #3

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Additional map: #2

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Additional map: #1

Fileemd_15101_additional_4.map
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Half map: #2

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Half map: #1

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Sample components

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Entire : DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D,...

EntireName: DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D, S561D and T567D)
Components
  • Complex: DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D, S561D and T567D)
    • Complex: FANCD2
      • Protein or peptide: FANCD2
    • Complex: FANCI3D
      • Protein or peptide: Fanconi anemia complementation group I
      • DNA: DNA (29-MER)
      • DNA: DNA (29-MER)

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Supramolecule #1: DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D,...

SupramoleculeName: DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI (S558D, S561D and T567D)
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 341 KDa

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Supramolecule #2: FANCD2

SupramoleculeName: FANCD2 / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: The 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.
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 164 KDa

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Supramolecule #3: FANCI3D

SupramoleculeName: FANCI3D / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Details: FANCI 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 ...Details: FANCI 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.
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: FANCD2

MacromoleculeName: FANCD2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 164.731344 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVSKRKLSKI DAAEESSKTD LQSRCPETKR SRISDKRAPS QGGLENEGVF EELLRTSGII LKVGEGQNEI AVDQTAFQKK LRVALEKHP SYPGVVNEFI SGLESHIKDR SQFKNCLLPC TPARTEGSRT LVHSYCESLI KLLLGIKILQ PAVVTLLLEK I PEFFFDVV ...String:
MVSKRKLSKI DAAEESSKTD LQSRCPETKR SRISDKRAPS QGGLENEGVF EELLRTSGII LKVGEGQNEI AVDQTAFQKK LRVALEKHP SYPGVVNEFI SGLESHIKDR SQFKNCLLPC TPARTEGSRT LVHSYCESLI KLLLGIKILQ PAVVTLLLEK I PEFFFDVV GTFGTNFPRL IVNQFKWLDG LLDSQDLVKK LMQMLSVSPV PIQHDIITSL PEILEDSQQN EVARELSCLL KQ GRRLTVP ILDALSRLDL DAELLAKVRQ SAMTIVPSVK LEDLPVVIKF ILHNVKAADA VEVISDLRKS LDLSSCVLPL QLL GSQRKL KSQAQASSSM SQVTTSQNCV KLLFDVIKLA VRFQKDVSEA WIKAIENSTS VSDHKVLDLI VLLLIHSTNS KNRK QTEKV LRSKIRLGCM PEQLMQNAFQ NHSMVIKDFF PSILSLAQTF LHSAHPAVVS FGSCMYKQAF AVFDSYCQQE VVCAL VTHV CSGNETELDI SLDVLTDLVI LHPSLLLRYA TFVKTILDSM QKLNPCQIRK LFYILSTLAF SQRQEGSYIQ DDMHMV IRK WLSSSVPNHK QMGIIGAVTM MGSVALKRNE ADGGLLERPE LSIECDGQLS TLLDLVGFCC EQTPEVLALY YDELANL IE KQKGNLDLQL LDKFGKSLVE DFPNDFVVDL SPTVDGSFLF PVKSLYNLDE DETQGAIAIN LLPLVSQSEP GRVADEMS N SRKRVVSPIC LSPCFRLLRL YTGEQNNGSL EEIDALLGCP LYLTDLEVEG KLDSLSKQER EFLCSLLFYA LNWFREVVN AFCQQQDAEM KGKVLTRLQN ITELQNVLGK CLAATPGYVP PPATFDSEAP EGVPSINAGG PVRKKNGKKR KSDSSKACSA ERTQADESS DGNQPDTELS ELEKSAAEKE TGNPLAQLQS YRPYFRELDL EVFSVLHCGL LTKSILDTEM HTEASEVVQL G PAELCFLL DDMCWKLEHV LTPGSTRRVP FLKERGNKDV GFSHLCQRSP KEVAVCVVKL LKPLCNHMEN MHNYFQTVIP NQ GVVDESG LNIQEYQLMS SCYHQLLLAF RLLFAWSGFS QHENSNLLRS ALQVLADRLK PGETEFLPLE ELISESFQYL LNF QASIPS FQCAFILTQV LMAISEKPMT GWKREKMASL AKQFLCQSWM KPGGDREKGS HFNSALHTLL CVYLEHTDNI LKAI EEISS VGVPELINSA KDGCSSTYPT LSRQTFPVFF RVMMAQLESS VKSIPAGKPS DSGEVQLEKL LKWNIAVRNF HILIN LVKV FDSRPVLSIC LKYGRLFVEA FLKLAMPLLD HSFKKHRDDV QSLLKTLQLS TRQLHHMCGH SKIHQDLGLT NHVPLL KKS LEQFVYRVKA MLAFNHCQEA FWVGVLKNRD LQGEEILSQA SAAPEEDSAE GSEEDTEDSA AEEPDGTDSD SGGAGRL EV LFQGPWSHPQ FEKGSAGSAA GSGAGWSHPQ FEK

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Macromolecule #2: Fanconi anemia complementation group I

MacromoleculeName: Fanconi anemia complementation group I / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 150.357156 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAQRILQLAA EGSPERLQEA LQGLTEGELG DMVTRQALRG RETAALLKGI FKGSPCSQQS GVLRRLQVYK HCVSLVESGD LHVGKVSEI IGLLMLEARQ LPGHALAELA TLFVEVIKRG SLSNGKSLEL FSTVLTALSN SKESLAYGKG ELNGEEFKKQ L INTLCSSK ...String:
MAQRILQLAA EGSPERLQEA LQGLTEGELG DMVTRQALRG RETAALLKGI FKGSPCSQQS GVLRRLQVYK HCVSLVESGD LHVGKVSEI IGLLMLEARQ LPGHALAELA TLFVEVIKRG SLSNGKSLEL FSTVLTALSN SKESLAYGKG ELNGEEFKKQ L INTLCSSK WDPQCVIHLA NMFRDIPLSG EELQFVVEKV LRMFSKLDLQ EIPPLVYQLL LLSAKGSKKT VLEGIISFFN QL DKRQKEE QRVPQSADLE VATVPLDQLR HVEGTVILHI VSAINLDQDI GEELIKHLKT EQQKDPGKAL CPFSVSLLLS TAV KHRLQE QIFDFLKTSI TRSCKDLQIL QASKFLQDLC PQQYDVTAVI LEVVKNSAFG WDHVTQGLVD LGFSLMESYE PKKS FGGKA AETNLGLSKM PAQQACKLGA SILLETFKVH EPIRSDILEQ VLNRVLTKAA SPVSHFIDLL SNIVVSAPLV LQNSS SRVT ETFDNLSFLP IDTVQGLLRA VQPLLKVSMS VRDSLILVLQ KAIFSRQLDA RKAAVAGFLL LLRNFKILGS LTSDQC DQA IGADQVQADV HACYNSAANE AFCLEILGSL RRCLSQQADV RLMLYEGFYD VLRRNSQLAS SIMETLLSQI KQYYLPQ QD LLPPLKLEGC IMAQGDQIFL QEPLAHLLCC IQHCLAWYKS TVHLCKGAED EEEEEDVGFE QNFEEMLESV TRRMIKSE L EDFELDKSAD FSPSSGVGVK NNIYAIQVMG ICEVLIEYNF KIGNFSKNKF EDVLGLFTCY NKLSEILKEK AGKNKSTLG NRIARSFLSM GFVSTLLTAL FRDNAQSHEE SLAVLRSSTE FMRYAVSVAL QKVQQLEEMG QTDGPDGQNP EKMFQNLCKI TRVLLWRYT SIPTAVEESG KKKGKSISLL CLEGLLRIFN TMQQLYAARI PQFLQALDIT DGDAEEADIN VTEKAAFQIR Q FQRSLVNQ LSSAEDDFNS KETQLLITIL STLSKLLDPG SQQFLQFLTW TVKICKENAL EDLSCCKGLL TLLFSLHVLY KS PVSLLRE LAQDIHACLG DIDQDVEIES RSHFAIVNVK TAAPTVCLLV LGQADKVLEE VDWLIKRLTI LGSDTSEDST QAS NQTQAL EKGVILQLGT LLTVFHELVQ TALPAGSCVD SLLRSLSKTY AILTSLIKHY IQACRSTSNT VPGRLEKLVK LSGS HLTPQ CYSFITYVQN IHSESLSFAE EKKKKKKEDE TAVVSTVMAK VLRDTKPIPN LIFAIEQYEK FLIHLSKKSK VNLMQ YMKL STSRDFRINA SMLDSVLQEQ NTEDAENEPD NNQSGTAEQP DENQEPQKKR RRKKHHHHHH

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Macromolecule #3: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.554697 KDa
SequenceString:
(DA)(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DC)(DT) (DC)(DT)(DA)(DG)(DA)(DC)(DA)(DG)(DC)(DT) (DG)(DC)(DT)(DC)(DA)(DG)(DG)(DA)(DT) (DT)(DG)(DA)(DT)(DC)(DT)(DG)(DT)(DA)(DA) (DT) (DG)(DG)(DC)(DC)

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Macromolecule #4: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.541715 KDa
SequenceString:
(DG)(DG)(DC)(DC)(DA)(DT)(DT)(DA)(DC)(DA) (DG)(DA)(DT)(DC)(DA)(DA)(DT)(DC)(DC)(DT) (DG)(DA)(DG)(DC)(DA)(DG)(DC)(DT)(DG) (DT)(DC)(DT)(DA)(DG)(DA)(DG)(DA)(DC)(DA) (DT) (DC)(DG)(DA)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMC8H18N2O4SHEPES
1.0 mMC9H15O6PTCEP
GridModel: UltrAuFoil R0.6/1 / Material: GOLD / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 14842 / Average exposure time: 1.5 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1356378
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: Ab initial model generated in RELION3.1
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 197436
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6tnf

chain_id: A

6tnf

chain_id: B
DetailsFor 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.
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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