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- EMDB-3476: Structure of FANCI-FANCD2 hetero-dimer co-expressed with FANCC-FA... -

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Basic information

Entry
Database: EMDB / ID: EMD-3476
TitleStructure of FANCI-FANCD2 hetero-dimer co-expressed with FANCC-FANCE-FANCF proteins.
Map data3D Reconstruction by negative stain EM of FANCI-FANCD2 co-expressed with FANCC-FANCE-FANCF.
Sample
  • Complex: Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly co-expressed with the FANCC-FANCE-FANCF ternary complex.
Methodsingle particle reconstruction / negative staining / Resolution: 18.3 Å
AuthorsSwuec P / Costa A
CitationJournal: Cell Rep / Year: 2017
Title: The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2.
Authors: Paolo Swuec / Ludovic Renault / Aaron Borg / Fenil Shah / Vincent J Murphy / Sylvie van Twest / Ambrosius P Snijders / Andrew J Deans / Alessandro Costa /
Abstract: Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core ...Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.
History
DepositionNov 10, 2016-
Header (metadata) releaseDec 14, 2016-
Map releaseDec 14, 2016-
UpdateJan 25, 2017-
Current statusJan 25, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3476.png

Downloads & links

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Map

FileDownload / File: emd_3476.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Reconstruction by negative stain EM of FANCI-FANCD2 co-expressed with FANCC-FANCE-FANCF.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.7 Å/pix.
x 128 pix.
= 345.6 Å		2.7 Å/pix.
x 128 pix.
= 345.6 Å		2.7 Å/pix.
x 128 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.66 / Movie #1: 0.066
Minimum - Maximum-0.07751421 - 0.1914667
Average (Standard dev.)0.000493103 (±0.012086637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0780.1910.000

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Supplemental data

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Sample components

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Entire : Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly...

EntireName: Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly co-expressed with the FANCC-FANCE-FANCF ternary complex.
Components
  • Complex: Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly co-expressed with the FANCC-FANCE-FANCF ternary complex.

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Supramolecule #1: Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly...

SupramoleculeName: Hetero-dimeric complex of FANCI and FANCD2 proteins recombinantly co-expressed with the FANCC-FANCE-FANCF ternary complex.
type: complex / ID: 1 / Parent: 0
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.005 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
120.0 mMNaClsodium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine
StainingType: NEGATIVE / Material: Uranyl formate
Details: The sample stained with a fresh 2% (wt/vol) uranyl formate solution.
GridModel: 3.05mm diameter, square mesh grids. / Material: COPPER / Mesh: 600 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 4.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Prior to sample incubation, the carbon-coated grid was glow-discharged for 30 seconds at 45 mA.

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Electron microscopy

MicroscopeJEOL 2100
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 447 / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: JEOL

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Image processing

Particle selectionNumber selected: 71295
CTF correctionSoftware: (Name: CTFFIND (ver. 3), RELION (ver. 1.4))
Startup modelType of model: INSILICO MODEL
In silico model: For three-dimensional reconstruction of the ID2 complex co-expressed with the CEF complex an initial 3D volume was generated using the e2inimodel.py program in the EMAN2 package. The ...In silico model: For three-dimensional reconstruction of the ID2 complex co-expressed with the CEF complex an initial 3D volume was generated using the e2inimodel.py program in the EMAN2 package. The initial volume was used as reference model for three-dimensional classification and refinement in RELION 1.4.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 13546
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)

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