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- EMDB-28863: PolyC9 in complex with aE11 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-28863
TitlePolyC9 in complex with aE11 Fab
Map dataLocal resolution filtered, sharpened combined map. Real-space cropped from box size 450px to 296px.
Sample
  • Complex: Quaternary complex of aE11 Fab and polyC9
    • Protein or peptide: Complement component 9
    • Protein or peptide: Monoclonal antibody aE11 Fab (heavy chain)
    • Protein or peptide: Monoclonal antibody aE11 Fab (light chain)
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBayly-Jones C
Funding support Australia, 2 items
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
DepositionNov 12, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJan 25, 2023-
Current statusJan 25, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28863.png

Downloads & links

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Map

FileDownload / File: emd_28863.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution filtered, sharpened combined map. Real-space cropped from box size 450px to 296px.
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.291
Minimum - Maximum-0.3509244 - 1.3200978
Average (Standard dev.)0.013754747 (±0.10956213)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin777792
Dimensions296296296
Spacing296296296
CellA=B=C: 414.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28863_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Unsharpened, local resolution filtered combined map. Real-space cropped...

Fileemd_28863_additional_1.map
AnnotationUnsharpened, local resolution filtered combined map. Real-space cropped from box size 450px to 296px.
Projections & Slices
AxesZYX

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Histogram

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Half map: Half map 1

Fileemd_28863_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_28863_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Quaternary complex of aE11 Fab and polyC9

EntireName: Quaternary complex of aE11 Fab and polyC9
Components
  • Complex: Quaternary complex of aE11 Fab and polyC9
    • Protein or peptide: Complement component 9
    • Protein or peptide: Monoclonal antibody aE11 Fab (heavy chain)
    • Protein or peptide: Monoclonal antibody aE11 Fab (light chain)

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Supramolecule #1: Quaternary complex of aE11 Fab and polyC9

SupramoleculeName: Quaternary complex of aE11 Fab and polyC9 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.6 MDa

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Macromolecule #1: Complement component 9

MacromoleculeName: Complement component 9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QYTTSYDPEL TESSGSASHI DCRMSPWSEW SQCDPCLRQM FRSRSIEVFG QFNGKRCTDA VGDRRQCVPT EPCEDAEDDC GNDFQCSTGR CIKMRLRCNG DNDCGDFSDE DDCESEPRPP CRDRVVEESE LARTAGYGIN ILGMDPLSTP FDNEFYNGLC NRDRDGNTLT ...String:
QYTTSYDPEL TESSGSASHI DCRMSPWSEW SQCDPCLRQM FRSRSIEVFG QFNGKRCTDA VGDRRQCVPT EPCEDAEDDC GNDFQCSTGR CIKMRLRCNG DNDCGDFSDE DDCESEPRPP CRDRVVEESE LARTAGYGIN ILGMDPLSTP FDNEFYNGLC NRDRDGNTLT YYRRPWNVAS LIYETKGEKN FRTEHYEEQI EAFKSIIQEK TSNFNAAISL KFTPTETNKA EQCCEETASS ISLHGKGSFR FSYSKNETYQ LFLSYSSKKE KMFLHVKGEI HLGRFVMRNR DVVLTTTFVD DIKALPTTYE KGEYFAFLET YGTHYSSSGS LGGLYELIYV LDKASMKRKG VELKDIKRCL GYHLDVSLAF SEISVGAEFN KDDCVKRGEG RAVNITSENL IDDVVSLIRG GTRKYAFELK EKLLRGTVID VTDFVNWASS INDAPVLISQ KLSPIYNLVP VKMKNAHLKK QNLERAIEDY INEFSVRKCH TCQNGGTVIL MDGKCLCACP FKFEGIACEI SKQKISEGLP ALEFPNEK

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Macromolecule #2: Monoclonal antibody aE11 Fab (heavy chain)

MacromoleculeName: Monoclonal antibody aE11 Fab (heavy chain) / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
VQLKESGPGL VAPSQSLSIT CTVSGFSLTV YGVNWIRQPP GKGLEWLGMI WGDGSTDYNS ALKSRLSITK DNSKSQVFLK MNSLQTDDTA RYYCARDRSY GGSSAWFGYW GQGTLVTVSA

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Macromolecule #3: Monoclonal antibody aE11 Fab (light chain)

MacromoleculeName: Monoclonal antibody aE11 Fab (light chain) / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
QMTQTTSSLS ASLGDRVTIS CRASHDISNY LNWYQQKPDG TLKLLIYYTS RLHSGVPSRF SGSGSGTDYS LTISNLEQED VATYFCQQGN YLPYTFGGGT KLEIK

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPEShepes buffer
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 0-40 / Average exposure time: 16.0 sec. / Average electron dose: 52.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 48248
Startup modelType of model: OTHER / Details: AB INITIO
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C22 (22 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 7783
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6dlw

,

3bae

)

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