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- EMDB-8141: Cryo-EM structure of the human FANCD2/FANCI complex reveals a nov... -

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Basic information

Entry
Database: EMDB / ID: EMD-8141
TitleCryo-EM structure of the human FANCD2/FANCI complex reveals a novel Tower domain required for FANCD2 monoubiquitination- Full length FANCD2/FANCI
Map dataNone
Sample
  • Complex: human FANCD2/FANCI complex
Biological speciesHelenium autumnale (sneezeweed)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.0 Å
AuthorsLiang CC / Li Z / Nicholson WV / Venien-Bryan C / Cohn MA
CitationJournal: Nat Commun / Year: 2016
Title: The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks before monoubiquitination of FANCD2.
Authors: Chih-Chao Liang / Zhuolun Li / David Lopez-Martinez / William V Nicholson / Catherine Vénien-Bryan / Martin A Cohn /
Abstract: The Fanconi anaemia (FA) pathway is important for the repair of DNA interstrand crosslinks (ICL). The FANCD2-FANCI complex is central to the pathway, and localizes to ICLs dependent on its ...The Fanconi anaemia (FA) pathway is important for the repair of DNA interstrand crosslinks (ICL). The FANCD2-FANCI complex is central to the pathway, and localizes to ICLs dependent on its monoubiquitination. It has remained elusive whether the complex is recruited before or after the critical monoubiquitination. Here, we report the first structural insight into the human FANCD2-FANCI complex by obtaining the cryo-EM structure. The complex contains an inner cavity, large enough to accommodate a double-stranded DNA helix, as well as a protruding Tower domain. Disease-causing mutations in the Tower domain are observed in several FA patients. Our work reveals that recruitment of the complex to a stalled replication fork serves as the trigger for the activating monoubiquitination event. Taken together, our results uncover the mechanism of how the FANCD2-FANCI complex activates the FA pathway, and explains the underlying molecular defect in FA patients with mutations in the Tower domain.
History
DepositionMar 31, 2016-
Header (metadata) releaseSep 7, 2016-
Map releaseSep 7, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.164
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.164
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8141.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 0.164 / Movie #1: 0.164
Minimum - Maximum-0.09978453 - 1.577032
Average (Standard dev.)0.0026387149 (±0.04330744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-68-68-68
Dimensions128128128
Spacing128128128
CellA=B=C: 448.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z448.000448.000448.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-41
NX/NY/NZ737384
MAP C/R/S123
start NC/NR/NS-68-68-68
NC/NR/NS128128128
D min/max/mean-0.1001.5770.003

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Supplemental data

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Sample components

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Entire : human FANCD2/FANCI complex

EntireName: human FANCD2/FANCI complex
Components
  • Complex: human FANCD2/FANCI complex

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Supramolecule #1: human FANCD2/FANCI complex

SupramoleculeName: human FANCD2/FANCI complex / type: complex / ID: 1 / Parent: 0
Details: FANCD2 is expressed using the pFastBac1 vector (Life Technologies) with an engineered N-terminal Flag-HA tag, and FANCI is expressed using the pFastBac1 vector (Life Technologies) with an ...Details: FANCD2 is expressed using the pFastBac1 vector (Life Technologies) with an engineered N-terminal Flag-HA tag, and FANCI is expressed using the pFastBac1 vector (Life Technologies) with an engineered N-terminal Flag-MBP tag or a N-terminal Flag-HA tag
Source (natural)Organism: Helenium autumnale (sneezeweed)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: hela / Recombinant plasmid: pX459
Molecular weightExperimental: 300 KDa

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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 / Component: (Name: Tris, KCl)
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber temperature: 293 K / Details: manually prepared with an home-made device.

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus min: 3.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 93.0 K
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 6 / Average exposure time: 1.0 sec. / Average electron dose: 10.0 e/Å2

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Image processing

Particle selectionNumber selected: 13222
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 8164 / Random conical tilt - Tilt angle: 50 degrees
Details: No initial model was existing, an initial model was calculated using the random conical tilt method implemented by SPIDER using 8164 particles negatively stained
Initial angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION / Number images used: 5058

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