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- EMDB-10293: Structure of the Fanconi Anaemia core complex (focussed map for b... -

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Basic information

Entry
Database: EMDB / ID: EMD-10293
TitleStructure of the Fanconi Anaemia core complex (focussed map for base region)
Map dataNone
Sample
  • Complex: Fanconi anaemia core complex
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsShakeel S / Rajendra E / Alcon P / He S / Scheres SHW / Passmore LA
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105192715 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structure of the Fanconi anaemia monoubiquitin ligase complex.
Authors: Shabih Shakeel / Eeson Rajendra / Pablo Alcón / Francis O'Reilly / Dror S Chorev / Sarah Maslen / Gianluca Degliesposti / Christopher J Russo / Shaoda He / Chris H Hill / J Mark Skehel / ...Authors: Shabih Shakeel / Eeson Rajendra / Pablo Alcón / Francis O'Reilly / Dror S Chorev / Sarah Maslen / Gianluca Degliesposti / Christopher J Russo / Shaoda He / Chris H Hill / J Mark Skehel / Sjors H W Scheres / Ketan J Patel / Juri Rappsilber / Carol V Robinson / Lori A Passmore /
Abstract: The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress. Genetic inactivation of this pathway by mutation ...The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.
History
DepositionSep 5, 2019-
Header (metadata) releaseNov 6, 2019-
Map releaseNov 6, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0128
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10293.png

Downloads & links

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Map

FileDownload / File: emd_10293.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 380 pix.
= 406.6 Å		1.07 Å/pix.
x 380 pix.
= 406.6 Å		1.07 Å/pix.
x 380 pix.
= 406.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0128 / Movie #1: 0.0128
Minimum - Maximum-0.020048974 - 0.044423144
Average (Standard dev.)0.0000685976 (±0.0011454074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-190-190-190
Dimensions380380380
Spacing380380380
CellA=B=C: 406.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z406.600406.600406.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-190-190-190
NC/NR/NS380380380
D min/max/mean-0.0200.0440.000

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Supplemental data

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Mask #1

Fileemd_10293_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: This is the map from auto refinement in Relion with T=5.

Fileemd_10293_additional.map
AnnotationThis is the map from auto refinement in Relion with T=5.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: This is the map from auto refinement in Relion with T=5.

Fileemd_10293_additional_1.map
AnnotationThis is the map from auto refinement in Relion with T=5.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: This is half1 map from auto refinement in Relion with T=5.

Fileemd_10293_half_map_1.map
AnnotationThis is half1 map from auto refinement in Relion with T=5.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: This is half2 map from auto refinement in Relion with T=5.

Fileemd_10293_half_map_2.map
AnnotationThis is half2 map from auto refinement in Relion with T=5.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fanconi anaemia core complex

EntireName: Fanconi anaemia core complex
Components
  • Complex: Fanconi anaemia core complex

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Supramolecule #1: Fanconi anaemia core complex

SupramoleculeName: Fanconi anaemia core complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#28
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 insect cells / Recombinant plasmid: pACEBac1
Molecular weightTheoretical: 860 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

Concentration0.7 mg/mL
BufferpH: 8 / Details: 50 mM HEPES pH 8.0, ~500 mM NaCl, 1 mM TCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time: 3 to 4.5 s Wait time: 0 s Drain time: 0 s Force: -10 N.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 4145 / Average electron dose: 40.0 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: -4.0 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 75000
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

CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: initial model generated in EMAN2.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: This is a focussed map for base region / Number images used: 169000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER

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