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- EMDB-15103: ubiquitinated DNA-bound FANCD2-FANCI3D -

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Basic information

Entry
Database: EMDB / ID: EMD-15103
Titleubiquitinated DNA-bound FANCD2-FANCI3D
Map data
Sample
  • Complex: ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI
    • Complex: FANCD2
      • Protein or peptide: FANCD2
    • Complex: FANCI3D
      • Protein or peptide: Fanconi anemia complementation group I
      • DNA: DNA chain S
      • DNA: DNA chain T
    • Complex: Polyubiquitin-C
      • Protein or peptide: Polyubiquitin-C
Keywordsnucleic acid protein complex / DNA clamp / solenoid domains / Fanconi anemia / DNA repair / DNA damage / DNA interstrand crosslink / DNA BINDING PROTEIN / ubiquitination
Biological speciesGallus gallus (chicken) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 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-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15103.png

Downloads & links

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Map

FileDownload / File: emd_15103.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 300 pix.
= 343.5 Å		1.15 Å/pix.
x 300 pix.
= 343.5 Å		1.15 Å/pix.
x 300 pix.
= 343.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.145 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0105
Minimum - Maximum-0.01876713 - 0.050311282
Average (Standard dev.)0.00008245763 (±0.0018030714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 343.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_15103_additional_1.map
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Additional map: #2

Fileemd_15103_additional_2.map
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Half map: #1

Fileemd_15103_half_map_1.map
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Half map: #2

Fileemd_15103_half_map_2.map
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Sample components

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Entire : ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic...

EntireName: ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI
Components
  • Complex: ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI
    • Complex: FANCD2
      • Protein or peptide: FANCD2
    • Complex: FANCI3D
      • Protein or peptide: Fanconi anemia complementation group I
      • DNA: DNA chain S
      • DNA: DNA chain T
    • Complex: Polyubiquitin-C
      • Protein or peptide: Polyubiquitin-C

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Supramolecule #1: ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic...

SupramoleculeName: ubiquitinated DNA-bound FANCD2-FANCI that contains phosphomimetic FANCI
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 349 KDa

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Supramolecule #2: FANCD2

SupramoleculeName: FANCD2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: FANCI3D

SupramoleculeName: FANCI3D / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4

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Supramolecule #4: Polyubiquitin-C

SupramoleculeName: Polyubiquitin-C / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5

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Macromolecule #1: FANCD2

MacromoleculeName: FANCD2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
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 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
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 #5: Polyubiquitin-C

MacromoleculeName: Polyubiquitin-C / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

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Macromolecule #3: DNA chain S

MacromoleculeName: DNA chain S / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
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 chain T

MacromoleculeName: DNA chain T / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
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
100.0 mMC3H4N2Imidazole
GridModel: UltrAuFoil R0.6/1 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 45 sec.
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 K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 6515 / Average exposure time: 7.8 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: 450882
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 25609
FSC plot (resolution estimation)

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