+Open data
-Basic information
Entry | Database: PDB / ID: 8de6 | |||||||||
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Title | Oligomeric C9 in complex with aE11 Fab | |||||||||
Components |
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Keywords | IMMUNE SYSTEM / Membrane Attack Complex / Complement Component 9 / polyC9 / aE11 | |||||||||
Function / homology | Function and homology information cell killing / Terminal pathway of complement / membrane attack complex / other organism cell membrane / complement activation, alternative pathway / complement activation / complement activation, classical pathway / Regulation of Complement cascade / protein homooligomerization / positive regulation of immune response ...cell killing / Terminal pathway of complement / membrane attack complex / other organism cell membrane / complement activation, alternative pathway / complement activation / complement activation, classical pathway / Regulation of Complement cascade / protein homooligomerization / positive regulation of immune response / killing of cells of another organism / blood microparticle / extracellular space / extracellular exosome / extracellular region / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) Mus musculus (house mouse) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Bayly-Jones, C. | |||||||||
Funding support | Australia, 2items
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Citation | Journal: Commun Biol / Year: 2023 Title: The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9. Authors: Charles Bayly-Jones / Bill H T Ho / Corinna Lau / Eleanor W W Leung / Laura D'Andrea / Christopher J Lupton / Susan M Ekkel / Hariprasad Venugopal / James C Whisstock / Tom E Mollnes / ...Authors: Charles Bayly-Jones / Bill H T Ho / Corinna Lau / Eleanor W W Leung / Laura D'Andrea / Christopher J Lupton / Susan M Ekkel / Hariprasad Venugopal / James C Whisstock / Tom E Mollnes / Bradley A Spicer / Michelle A Dunstone / Abstract: The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is ...The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8de6.cif.gz | 336.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8de6.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8de6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8de6_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8de6_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8de6_validation.xml.gz | 58.7 KB | Display | |
Data in CIF | 8de6_validation.cif.gz | 87.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/de/8de6 ftp://data.pdbj.org/pub/pdb/validation_reports/de/8de6 | HTTPS FTP |
-Related structure data
Related structure data | 27385MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Antibody , 2 types, 4 molecules EBFI
#1: Antibody | Mass: 15219.310 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: Papain-treated IgG / Source: (natural) Mus musculus (house mouse) / Plasmid details: Hybridoma #2: Antibody | Mass: 13927.579 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: Papain-digested IgG / Source: (natural) Mus musculus (house mouse) / Plasmid details: Hybridoma |
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-Protein / Non-polymers , 2 types, 6 molecules ACG
#3: Protein | Mass: 63836.809 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Cell line: Hepatocytes / Gene: C9 / Cell line (production host): HEK293 Expi / Production host: Homo sapiens (human) / References: UniProt: P02748 #6: Chemical | |
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-Sugars , 2 types, 9 molecules
#4: Sugar | #5: Sugar | ChemComp-BMA / |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Quaternary complex of aE11 Fab and polyC9 / Type: COMPLEX Details: Fab fragment of aE11 generated by proteolytic cleavage of aE11 IgG antibody, in complex with recombinant human C9 incubated at 37 degrees Celsius to form homo-oligomeric C9. Entity ID: #1-#3 / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 2.6 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | |||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) / Cell: HEK293 Expi / Plasmid: pSecTag | |||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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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 defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 16 sec. / Electron dose: 52.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
Image scans | Movie frames/image: 40 / Used frames/image: 0-40 |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 48248 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C22 (22 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10061 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 163 / Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||
Atomic model building |
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