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- PDB-6srs: Structure of the Fanconi anaemia core subcomplex -

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Basic information

Entry
Database: PDB / ID: 6srs
TitleStructure of the Fanconi anaemia core subcomplex
Components
  • (Unassigned secondary structure elements (central region, proposed FANCB- ...) x 11
  • (Unassigned secondary structure elements (proposed ...) x 6
  • Fanconi anaemia protein FANCLFanconi anemia
KeywordsLIGASE / Fanconi Anaemia core complex / E3 ligase / DNA repair
Function / homology
Function and homology information


Fanconi Anemia Pathway in DNA repair / Fanconi Anemia Pathway / Fanconi anaemia nuclear complex / protein monoubiquitination / interstrand cross-link repair / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / DNA repair / DNA damage response / nucleoplasm ...Fanconi Anemia Pathway in DNA repair / Fanconi Anemia Pathway / Fanconi anaemia nuclear complex / protein monoubiquitination / interstrand cross-link repair / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / DNA repair / DNA damage response / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
FANCL, UBC-like domain 2 / FANCL, UBC-like domain 3 / Fanconi anemia complex, subunit FancL, WD-repeat containing domain / E3 ubiquitin-protein ligase FANCL / FANCL C-terminal domain / FANCL, UBC-like domain 3 superfamily / FANCL UBC-like domain 1 / FANCL C-terminal domain / FANCL UBC-like domain 2 / FANCL UBC-like domain 3 ...FANCL, UBC-like domain 2 / FANCL, UBC-like domain 3 / Fanconi anemia complex, subunit FancL, WD-repeat containing domain / E3 ubiquitin-protein ligase FANCL / FANCL C-terminal domain / FANCL, UBC-like domain 3 superfamily / FANCL UBC-like domain 1 / FANCL C-terminal domain / FANCL UBC-like domain 2 / FANCL UBC-like domain 3 / Ubiquitin-conjugating enzyme/RWD-like / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Fanconi anemia complementation group L
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsShakeel, S. / Rajendra, E. / Alcon, P. / He, S. / Scheres, S.H.W. / Passmore, L.A.
Funding support United Kingdom, 1items
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 20, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
B: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
C: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
D: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
E: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
F: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
G: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
H: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
I: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
J: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
K: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
L: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
a: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
b: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
c: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
d: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
e: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
f: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
g: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
h: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
i: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
j: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
k: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
l: Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)
M: Unassigned secondary structure elements (proposed FANCB)
N: Unassigned secondary structure elements (proposed FAAP100)
O: Unassigned secondary structure elements (proposed FANCB)
P: Unassigned secondary structure elements (proposed FAAP100)
R: Unassigned secondary structure elements (proposed FANCG)
S: Unassigned secondary structure elements (proposed FANCG)
Q: Fanconi anaemia protein FANCL
m: Unassigned secondary structure elements (proposed FANCB)
n: Unassigned secondary structure elements (proposed FAAP100)
o: Unassigned secondary structure elements (proposed FANCB)
p: Unassigned secondary structure elements (proposed FAAP100)
r: Unassigned secondary structure elements (proposed FANCG)
s: Unassigned secondary structure elements (proposed FANCG)
q: Fanconi anaemia protein FANCL


Theoretical massNumber of molelcules
Total (without water)270,44538
Polymers270,44538
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Unassigned secondary structure elements (central region, proposed FANCB- ... , 11 types, 24 molecules AaBbCcDdEeFfGgHJhjIiKkLl

#1: Protein Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 6485.986 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#2: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 2145.636 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#3: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 2571.161 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#4: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 1975.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 783.958 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#6: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 1294.587 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#7: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 2230.741 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#8: Protein/peptide
Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 1464.797 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#9: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 3166.895 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#10: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 1805.216 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#11: Protein/peptide Unassigned secondary structure elements (central region, proposed FANCB-FAAP100)


Mass: 1549.902 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)

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Unassigned secondary structure elements (proposed ... , 6 types, 12 molecules MmNnOoPpRrSs

#12: Protein Unassigned secondary structure elements (proposed FANCB)


Mass: 10230.603 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#13: Protein/peptide Unassigned secondary structure elements (proposed FAAP100)


Mass: 3677.524 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#14: Protein Unassigned secondary structure elements (proposed FANCB)


Mass: 23762.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#15: Protein Unassigned secondary structure elements (proposed FAAP100)


Mass: 23506.855 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#16: Protein Unassigned secondary structure elements (proposed FANCG)


Mass: 24272.791 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)
#17: Protein Unassigned secondary structure elements (proposed FANCG)


Mass: 12358.226 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Production host: Spodoptera frugiperda (fall armyworm)

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Protein , 1 types, 2 molecules Qq

#18: Protein Fanconi anaemia protein FANCL / Fanconi anemia


Mass: 10475.292 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: FANCL / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3MUH5

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Fanconi anaemia core subcomplex / Type: COMPLEX / Entity ID: #1, #10-#18, #2-#9 / Source: RECOMBINANT
Molecular weightValue: 0.86 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9 insect cells / Plasmid: pACEBac1
Buffer solutionpH: 8 / Details: 50 mM HEPES pH 8.0, ~500 mM NaCl, 1 mM TCEP
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: -4000 nm / Nominal defocus min: -1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 4145

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
9REFMACmodel refinement
10PHENIXmodel refinement
11Cootmodel refinement
12RELION3initial Euler assignment
13RELION3final Euler assignment
14RELION3classification
15RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49423
Details: Local symmetry was applied for two regions followed by autorefinement was was done with tau_fudge (T)=100. Map was post processed to obtain realistic resolution for the map.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER

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