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- PDB-8de6: Oligomeric C9 in complex with aE11 Fab -

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Basic information

Entry
Database: PDB / ID: 8de6
TitleOligomeric C9 in complex with aE11 Fab
Components
  • Complement component C9
  • aE11 Fab VH
  • aE11 Fab VL
KeywordsIMMUNE 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
Membrane attack complex component/perforin/complement C9 / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / Thrombospondin type 1 domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site ...Membrane attack complex component/perforin/complement C9 / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / Thrombospondin type 1 domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / Thrombospondin type-1 (TSP1) repeat superfamily / LDL-receptor class A (LDLRA) domain profile. / Thrombospondin type-1 (TSP1) repeat profile. / Thrombospondin type 1 repeats / Thrombospondin type-1 (TSP1) repeat / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain signature 1.
Similarity search - Domain/homology
beta-D-mannopyranose / Complement component C9
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBayly-Jones, C.
Funding support Australia, 2items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP180100040 Australia
Australian Research Council (ARC)FT150100049 Australia
CitationJournal: 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
DepositionJun 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: aE11 Fab VH
F: aE11 Fab VL
A: Complement component C9
B: aE11 Fab VH
I: aE11 Fab VL
C: Complement component C9
G: Complement component C9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,66919
Polymers249,8047
Non-polymers1,86512
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 4 molecules EBFI

#1: Antibody aE11 Fab VH


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 aE11 Fab VL


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 Complement component C9


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 ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca

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Sugars , 2 types, 9 molecules

#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Sugar
ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-mannopyranoseCOMMON NAMEGMML 1.0
b-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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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: 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
Molecular weightValue: 2.6 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 Expi / Plasmid: pSecTag
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMhepes bufferHEPES1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 52.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40 / Used frames/image: 0-40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 48248
SymmetryPoint symmetry: C22 (22 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10061 / Symmetry type: POINT
Atomic model buildingB value: 163 / Protocol: FLEXIBLE FIT
Atomic model building
IDPDB-ID 3D fitting-ID
16DLW

6dlw

1
23BAE

3bae

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0113501
ELECTRON MICROSCOPYf_angle_d0.8818225
ELECTRON MICROSCOPYf_dihedral_angle_d10.7235068
ELECTRON MICROSCOPYf_chiral_restr0.0581980
ELECTRON MICROSCOPYf_plane_restr0.0072359

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