+Open data
-Basic information
Entry | Database: PDB / ID: 6srs | ||||||
---|---|---|---|---|---|---|---|
Title | Structure of the Fanconi anaemia core subcomplex | ||||||
Components |
| ||||||
Keywords | LIGASE / Fanconi Anaemia core complex / E3 ligase / DNA repair | ||||||
Function / homology | Function and homology information PKR-mediated signaling / 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 ...PKR-mediated signaling / 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 / nucleus Similarity search - Function | ||||||
Biological species | Gallus gallus (chicken) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å | ||||||
Authors | Shakeel, S. / Rajendra, E. / Alcon, P. / He, S. / Scheres, S.H.W. / Passmore, L.A. | ||||||
Funding support | United Kingdom, 1items
| ||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6srs.cif.gz | 287 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb6srs.ent.gz | 232.4 KB | Display | PDB format |
PDBx/mmJSON format | 6srs.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6srs_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 6srs_full_validation.pdf.gz | 994.3 KB | Display | |
Data in XML | 6srs_validation.xml.gz | 54.1 KB | Display | |
Data in CIF | 6srs_validation.cif.gz | 85.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sr/6srs ftp://data.pdbj.org/pub/pdb/validation_reports/sr/6srs | HTTPS FTP |
-Related structure data
Related structure data | 10294MC 6sriC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
-Unassigned secondary structure elements (central region, proposed FANCB- ... , 11 types, 24 molecules AaBbCcDdEeFfGgHJhjIiKkLl
#1: Protein | 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 | 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 | 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 | 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 | 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 | 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 | 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 | 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 | 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 | 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 | 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) |
---|
-Unassigned secondary structure elements (proposed ... , 6 types, 12 molecules MmNnOoPpRrSs
#12: Protein | 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 | 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 | 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 | 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 | 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 | 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) |
---|
-Protein , 1 types, 2 molecules Qq
#18: Protein | 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 |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Fanconi anaemia core subcomplex / Type: COMPLEX / Entity ID: #1, #10-#18, #2-#9 / Source: RECOMBINANT |
---|---|
Molecular weight | Value: 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 solution | pH: 8 / Details: 50 mM HEPES pH 8.0, ~500 mM NaCl, 1 mM TCEP |
Specimen | Conc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3 |
Vitrification | Instrument: 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 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 4145 |
-Processing
EM software |
| ||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: OTHER |