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- EMDB-10534: Structure of FANCD2 homodimer -

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Basic information

Entry
Database: EMDB / ID: EMD-10534
TitleStructure of FANCD2 homodimer
Map dataPost processed map from Relion
Sample
  • Complex: FANCD2 homodimer
    • Protein or peptide: Uncharacterized protein
KeywordsFanconi anaemia / ubiquitin / DNA repair / DNA damage / inter-strand crosslink / DNA BINDING PROTEIN
Function / homology
Function and homology information


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 / nucleus
Similarity search - Function
Fanconi anaemia protein FANCD2 / Fanconi anaemia protein FancD2 nuclease
Similarity search - Domain/homology
Fanconi anemia complementation group D2
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAlcon P / Shakeel S
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105192715 United Kingdom
European Molecular Biology OrganizationALTF 692-2018 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair.
Authors: Pablo Alcón / Shabih Shakeel / Zhuo A Chen / Juri Rappsilber / Ketan J Patel / Lori A Passmore /
Abstract: Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause ...Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.
History
DepositionDec 8, 2019-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tni
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10534.png

Downloads & links

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Map

FileDownload / File: emd_10534.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost processed map from Relion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 450 pix.
= 477. Å		1.06 Å/pix.
x 450 pix.
= 477. Å		1.06 Å/pix.
x 450 pix.
= 477. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0134 / Movie #1: 0.0134
Minimum - Maximum-0.06207513 - 0.09728522
Average (Standard dev.)0.000033782577 (±0.0009958955)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 476.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z477.000477.000477.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS450450450
D min/max/mean-0.0620.0970.000

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Supplemental data

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Additional map: Map from auto-refinement in Relion

Fileemd_10534_additional.map
AnnotationMap from auto-refinement in Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1 map from auto-refinement in Relion

Fileemd_10534_half_map_1.map
AnnotationHalf1 map from auto-refinement in Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2 map from auto-refinement in Relion

Fileemd_10534_half_map_2.map
AnnotationHalf2 map from auto-refinement in Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FANCD2 homodimer

EntireName: FANCD2 homodimer
Components
  • Complex: FANCD2 homodimer
    • Protein or peptide: Uncharacterized protein

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Supramolecule #1: FANCD2 homodimer

SupramoleculeName: FANCD2 homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Uncharacterized protein

MacromoleculeName: Uncharacterized protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / 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

UniProtKB: Fanconi anemia complementation group D2

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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

Concentration0.3 mg/mL
BufferpH: 8
Details: 50 mM HEPES, 100 mM imidazole, 150 mM NaCl, 1 mM TCEP
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300
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
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3000000
Startup modelType of model: OTHER
Details: Random model generated by stochastic gradient algorithm
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 901085
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3s4w


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6tni:
Structure of FANCD2 homodimer

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