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- EMDB-27385: Oligomeric C9 in complex with aE11 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-27385
TitleOligomeric C9 in complex with aE11 Fab
Map dataSharpened filtered map
Sample
  • Complex: Quaternary complex of aE11 Fab and polyC9
    • Protein or peptide: aE11 Fab VH
    • Protein or peptide: aE11 Fab VL
    • Protein or peptide: Complement component C9
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: beta-D-mannopyranose
  • Ligand: CALCIUM IONCalcium
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 / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. ...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 / 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 domain / LDL-receptor class A (LDLRA) domain profile. / Thrombospondin type-1 (TSP1) repeat superfamily / 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
Complement component C9
Similarity search - Component
Biological speciesHomo sapiens (human) / mouse (mice)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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
DepositionJun 20, 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_27385.png

Downloads & links

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Map

FileDownload / File: emd_27385.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened filtered map
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.893
Minimum - Maximum-3.492268 - 6.0409937
Average (Standard dev.)0.0005079184 (±0.14423886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27385_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened filtered map

Fileemd_27385_additional_1.map
AnnotationUnsharpened filtered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A, unfiltered

Fileemd_27385_half_map_1.map
AnnotationHalf-map A, unfiltered
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B, unfiltered

Fileemd_27385_half_map_2.map
AnnotationHalf-map B, unfiltered
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: aE11 Fab VH
    • Protein or peptide: aE11 Fab VL
    • Protein or peptide: Complement component C9
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: beta-D-mannopyranose
  • Ligand: CALCIUM IONCalcium

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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: #1-#3
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: aE11 Fab VH

MacromoleculeName: aE11 Fab VH / type: protein_or_peptide / ID: 1 / Details: Papain-treated IgG / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: mouse (mice)
Molecular weightTheoretical: 15.21931 KDa
SequenceString:
MAVLALLFCL VTFPSCILSQ VQLKESGPGL VAPSQSLSIT CTVSGFSLTV YGVNWIRQPP GKGLEWLGMI WGDGSTDYNS ALKSRLSIT KDNSKSQVFL KMNSLQTDDT ARYYCARDRS YGGSSAWFGY WGQGTLVTVS A

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Macromolecule #2: aE11 Fab VL

MacromoleculeName: aE11 Fab VL / type: protein_or_peptide / ID: 2 / Details: Papain-digested IgG / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: mouse (mice)
Molecular weightTheoretical: 13.927579 KDa
SequenceString:
MSSAQFLGLL LLCFQGTRCD IQMTQTTSSL SASLGDRVTI SCRASHDISN YLNWYQQKPD GTLKLLIYYT SRLHSGVPSR FSGSGSGTD YSLTISNLEQ EDVATYFCQQ GNYLPYTFGG GTKLEIK

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Macromolecule #3: Complement component C9

MacromoleculeName: Complement component C9 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.836809 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METDTLLLWV LLLWVPGSTG DAAQPAQYTT SYDPELTESS GSASHIDCRM SPWSEWSQCD PCLRQMFRSR SIEVFGQFNG KRCTDAVGD RRQCVPTEPC EDAEDDCGND FQCSTGRCIK MRLRCNGDND CGDFSDEDDC ESEPRPPCRD RVVEESELAR T AGYGINIL ...String:
METDTLLLWV LLLWVPGSTG DAAQPAQYTT SYDPELTESS GSASHIDCRM SPWSEWSQCD PCLRQMFRSR SIEVFGQFNG KRCTDAVGD RRQCVPTEPC EDAEDDCGND FQCSTGRCIK MRLRCNGDND CGDFSDEDDC ESEPRPPCRD RVVEESELAR T AGYGINIL GMDPLSTPFD NEFYNGLCNR DRDGNTLTYY RRPWNVASLI YETKGEKNFR TEHYEEQIEA FKSIIQEKTS NF NAAISLK FTPTETNKAE QCCEETASSI SLHGKGSFRF SYSKNETYQL FLSYSSKKEK MFLHVKGEIH LGRFVMRNRD VVL TTTFVD DIKALPTTYE KGEYFAFLET YGTHYSSSGS LGGLYELIYV LDKASMKRKG VELKDIKRCL GYHLDVSLAF SEIS VGAEF NKDDCVKRGE GRAVNITSEN LIDDVVSLIR GGTRKYAFEL KEKLLRGTVI DVTDFVNWAS SINDAPVLIS QKLSP IYNL VPVKMKNAHL KKQNLERAIE DYINEFSVRK CHTCQNGGTV ILMDGKCLCA CPFKFEGIAC EISKQKISEG LPALEF PNE K

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: beta-D-mannopyranose

MacromoleculeName: beta-D-mannopyranose / type: ligand / ID: 5 / Number of copies: 6 / Formula: BMA
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BMA:
beta-D-mannopyranose / Mannose

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
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 reconstructionApplied symmetry - Point group: C22 (22 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 10061
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6dlw

,

3bae

)
RefinementProtocol: FLEXIBLE FIT / Overall B value: 163
Output model

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

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