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- EMDB-10844: Mono-ubiquitinated FANCD2 in complex with mono-ubiquitinated FANC... -

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Basic information

Entry
Database: EMDB / ID: EMD-10844
TitleMono-ubiquitinated FANCD2 in complex with mono-ubiquitinated FANCI and dsDNA
Map dataMono-ubiquitinated FANCI and mono-ubiquitinated FANCD2 in complex with double stranded DNA
Sample
  • Complex: Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI, and 32 base pair dsDNA
    • Complex: Mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI
      • Protein or peptide: FANCD2 mono-ubiquitinated at K561
      • Protein or peptide: FANCI mono-ubiquitinated at K523
    • Complex: 32 base pair dsDNA
      • DNA: double stranded DNA, 32 base pairs
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsRennie ML / Lemonidis K / Arkinson C / Chaugule VK / Clarke M / Streetley J / Spagnolo L / Walden H
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-2015-CoG-681582 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_PC_17135 United Kingdom
CitationJournal: EMBO Rep / Year: 2020
Title: Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA.
Authors: Martin L Rennie / Kimon Lemonidis / Connor Arkinson / Viduth K Chaugule / Mairi Clarke / James Streetley / Laura Spagnolo / Helen Walden /
Abstract: The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the ...The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.
History
DepositionApr 8, 2020-
Header (metadata) releaseJun 17, 2020-
Map releaseJun 17, 2020-
UpdateJul 15, 2020-
Current statusJul 15, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10844.png

Downloads & links

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Map

FileDownload / File: emd_10844.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMono-ubiquitinated FANCI and mono-ubiquitinated FANCD2 in complex with double stranded DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 277.76 Å		1.09 Å/pix.
x 256 pix.
= 277.76 Å		1.09 Å/pix.
x 256 pix.
= 277.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.085 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.011 / Movie #1: 0.011
Minimum - Maximum-0.007672439 - 0.03029835
Average (Standard dev.)0.00039954696 (±0.0026101521)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 277.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z277.760277.760277.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0080.0300.000

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Supplemental data

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Sample components

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Entire : Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated ...

EntireName: Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI, and 32 base pair dsDNA
Components
  • Complex: Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI, and 32 base pair dsDNA
    • Complex: Mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI
      • Protein or peptide: FANCD2 mono-ubiquitinated at K561
      • Protein or peptide: FANCI mono-ubiquitinated at K523
    • Complex: 32 base pair dsDNA
      • DNA: double stranded DNA, 32 base pairs

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Supramolecule #1: Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated ...

SupramoleculeName: Ternary complex of mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI, and 32 base pair dsDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 350 KDa

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Supramolecule #2: Mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI

SupramoleculeName: Mono-ubiquitinated FANCD2, mono-ubiquitinated FANCI / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: 32 base pair dsDNA

SupramoleculeName: 32 base pair dsDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: FANCD2 mono-ubiquitinated at K561

MacromoleculeName: FANCD2 mono-ubiquitinated at K561 / type: protein_or_peptide / ID: 1
Details: Additional single ubiquitin conjugated to K561 (ubiquitin sequence: GPGSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG)
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GPGSMVSKRR LSKSEDKESL TEDASKTRKQ PLSKKTKKSH IANEVEENDS IFVKLL KIS GIILKTGESQ NQLAVDQIAF QKKLFQTL R RHPSYPKIIE EFVSGLESYI EDEDSFRNCL LSCERLQDE EASMGASYSK SLIKLLLGID ILQPAIIKTL FEKLPEYFFE ...String:
GPGSMVSKRR LSKSEDKESL TEDASKTRKQ PLSKKTKKSH IANEVEENDS IFVKLL KIS GIILKTGESQ NQLAVDQIAF QKKLFQTL R RHPSYPKIIE EFVSGLESYI EDEDSFRNCL LSCERLQDE EASMGASYSK SLIKLLLGID ILQPAIIKTL FEKLPEYFFE NKNSDEINIP RLIVSQLKWL DRVVDGKDL TTKI MQLIS IAPENLQHDI ITSLPEILGD SQHADVGKEL SDLLIENTSL TVPILDVLSS LRLDPNFLLK VRQLVMD KL SSIRLEDLPV IIK FILHSV TAMDTLEVIS ELREKLDLQH CVLPSRLQAS QVKLKSKGRA SSSGNQESS GQSCIILLFD VIKSAIRYEK TISEAWIKAI ENTASVSEHK VFDLV MLFI IYSTNTQTKK YIDRVLRNKI RSGCI QEQL LQSTFSVHYL VLKDMCSSIL SLAQSLLHSL DQSIISFGSL LYKYAFKFFD TYCQQEVVGA LVTHICS GN EAEVDTALDV LLELVVLNPS AMMMNAVFVK GILDYLDNIS PQQIRKLFYV LSTLAFSKQN EASSHIQD D MHLVIRKQLS STVFKYKLIG IIGAVTMAG IMAADRSESP SLTQERANLS DEQCTQVTSL LQLVHSCSE QSPQASALYY DEFANLIQHE KLDPKALEWV GHTICNDFQD AFVVDSCVVP EGDFPFPVKA L YGLEEYD TQDGIAINLL PLLFSQDFAK DGGPVTSQES GQKLVSPLCL APYFRLLRLC VERQHNGNLE EIDGLLDCPI FLTDLEPGE KLESMSAKER SFMC SLIFL TLNWFREIVN AFCQETSPEM KGKVLTRLKH IVELQIILEK YLA VTPDYV PPLGNFDVET LDITPHTVTA ISAKIRKKGK IERKQKTDGS KTSSSD TLS EEKNSECDPT PSHRG QLNK EFTGKEEKTS LLLHNSHAFF RELDIEVFSI LHCGLVTKFI LDTEMHTEAT EVVQLGPPEL LFLLEDL SQ KLESMLTPP IARRVPFLKN KGSRNIGFSH LQQRSAQEIV HCVFQLLTPM CNHLENIHNY FQCLAAEN H GVVDGPGVKV QEYHIMSSCY QRLLQIFHGL FAWSGFSQPE N QNLLYSAL HVLSSRLKQG EHSQPLE EL LSQSVHYLQN FHQSIPSFQC ALYLIRLLMV ILEKSTASAQ NKEKIASLAR QFLCRVWPSG DKEKSNIS N DQLH ALLCI YLEHTESILK AIEEIAGVGV PELINSPKDA SSSTFPTLTR HTFVVFFRVM MAELEKTVKK IE PGTAADS QQIHEEKLLY WNMAVRDFSI LINLIK VFD SHPVLHVCLK YGRLFVEAFL KQCMPLLDFS FRK HREDVL SLLETFQLDT RLLHHLCGHS KIHQDTRLTQ HVPLLKKTLE LLVCRVKAML TLNNCREA F WLGN LKNRD LQGEEIKSQN SQESTADESE DDMSSQASKS KATEDGEEDE VSAGEKEQDS DESYDDSD

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Macromolecule #2: FANCI mono-ubiquitinated at K523

MacromoleculeName: FANCI mono-ubiquitinated at K523 / type: protein_or_peptide / ID: 2
Details: Additional single ubiquitin conjugated to K523 (ubiquitin sequence: GPGSMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG)
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHENL YFQGKPIPNP LLGLDSTMDQ KILSLAAEKT ADKLQEFLQT LREGDLTNLL QNQAVKGKV AGALLRAIFK GSPCSEEAGT LRRRKIYTCC I QLVESGDL QKEIASEIIG LLMLEAHHFP GPLLVELANE FIS AVREGS LVNGKSLELL PIILTALATK ...String:
MHHHHHHENL YFQGKPIPNP LLGLDSTMDQ KILSLAAEKT ADKLQEFLQT LREGDLTNLL QNQAVKGKV AGALLRAIFK GSPCSEEAGT LRRRKIYTCC I QLVESGDL QKEIASEIIG LLMLEAHHFP GPLLVELANE FIS AVREGS LVNGKSLELL PIILTALATK KENLAYGKGV LSGEECKKQL INTLCSGRWD QQY VIQLTS MFKD VPLTA EEVEFVVEKA LSMFSKMNLQ EIPPLVYQLL VLSSKGSRKS VLEGIIAFFS ALDKQHNEEQ SGDEL LDVV TVPSGELRHV EGTIIL HIV FAIKLDYELG RELVKHLKVG QQGDSNNNLS PFSIALLLSV TRIQRFQD Q VLDLLKTSVV KSFKDLQLLQ GSKFLQNLVP HRSYVSTMIL EVVKNSVH S WDHVTQGLVE LGFILMDS Y GPKKVLDGKT IETSPSLSRM PNQHACKLGA NILLETFKIH EMIRQEILEQ VLNRVVTRAS SPISHFLDLL SNIVMYAPL V LQSCSSKV TEAFDYLSFL PLQTVQRLLK AVQPLLKVSM SMRDCLILVL RKAMFANQLD A RKSAVAGF LLLLKNFKVL GSLSSSQCSQ SLSVSQVHVD VHS HYNSVA NETFCLEIMD SLRRCLSQQA D VRLMLYEG FYDVLRRNSQ LANSVMQTLL SQLKQFYEPK PDLLPPLKLE ACILTQGDKI SLQEPLDYLL CC IQH CLA WYKNTVIPLQ QGEEEEEEEE AFYEDLDDIL ESITNRMIKS ELEDFELDKS ADFSQSTSIG IKNNI CAFL VMGVCEVLIE YNFSISSFSK NRFEDILS L FMCYKKLSDI LNEKAGKAKT KMANKTSDSL LSMKFVS SL LTALFRDSIQ SHQESLSVLR SSNEFMRYAV NVALQKVQQL KETGHVSGPD GQNPEKIFQN LCDITR VL LWRYTSIPTS VEESGKKEKG KSISLLCLEG LQKIFSAVQQ FYQPKIQQFL RALDVTDKEG EEREDADV S VTQRTAFQIR QFQRSLLNLL SSQ EEDFNS KEALLLVTVL TSLSKLLEPS SPQFVQMLSW TSKICKENS REDALFCKSL MNLLFSLHVS YKSPVILLRD LSQDIHGHLG DIDQDVEVEK TNHFA IVNL RTAAPTVCLL V LSQAEKVL EEVDWLITKL KGQVSQETLS EEASSQATLP NQPVEKAIIM QLGTLLTFFH ELVQTALPSG SC VDTLLKD LCKMYTTL T ALVRYYLQVC QSSGGIPKNM EKLVKLSGSH LTPLCYSFIS YVQNKSKSLN YTG EKKEKP AAVATAMARV LRETKPIPNL IFAIEQYEKF LIHLSKKSKV NLMQHMKLS TSRDFKIKGN ILDMVLREDG EDENEEGTAS EHGGQNKEPA KKKRKK

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Macromolecule #3: double stranded DNA, 32 base pairs

MacromoleculeName: double stranded DNA, 32 base pairs / type: dna / ID: 3
Details: Sense strand (anti-sense strand: CGATCGGTAACGTATGCTGAATCTGGTGCTGG)
Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CCAGCACCAG ATTCAGCATA CGTTACCGAT CG

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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

Concentration1.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris/HCl pH 8.0
100.0 mMNaClsodium chloride
2.0 mMC4H10O2S2DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.55 K / Instrument: FEI VITROBOT MARK IV
DetailsComplex components were mixed then sample was exchanged into cryoEM buffer via a desalting column

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsMicroscope access provided by eBIC (Diamond Light Source) under proposal EM16637.
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 44.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Stochastic gradient descent implemented in RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12710
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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