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- EMDB-3135: Electron cryo-microscopy of an immune pore -

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Basic information

Entry
Database: EMDB / ID: EMD-3135
TitleElectron cryo-microscopy of an immune pore
Map dataReconstruction of the barrel of the membrane attack complex
Sample
  • Sample: Membrane attack complexComplement membrane attack complex
  • Protein or peptide: C5
  • Protein or peptide: C6
  • Protein or peptide: C7
  • Protein or peptide: C8 alpha
  • Protein or peptide: C8 beta
  • Protein or peptide: C8 gamma
  • Protein or peptide: C9
Keywordscryo-EM / single particles / membrane protein
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsSerna M / Bubeck D
CitationJournal: Nat Commun / Year: 2016
Title: Structural basis of complement membrane attack complex formation.
Authors: Marina Serna / Joanna L Giles / B Paul Morgan / Doryen Bubeck /
Abstract: In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; ...In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a 'split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.
History
DepositionAug 25, 2015-
Header (metadata) releaseJan 13, 2016-
Map releaseFeb 10, 2016-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD3135_imag...

Downloads & links

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Map

FileDownload / File: emd_3135.map.gz / Format: CCP4 / Size: 210.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the barrel of the membrane attack complex
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.08353171 - 0.24291411
Average (Standard dev.)0.00097472 (±0.00883864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z537.600537.600537.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0840.2430.001

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Supplemental data

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Sample components

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Entire : Membrane attack complex

EntireName: Membrane attack complexComplement membrane attack complex
Components
  • Sample: Membrane attack complexComplement membrane attack complex
  • Protein or peptide: C5
  • Protein or peptide: C6
  • Protein or peptide: C7
  • Protein or peptide: C8 alpha
  • Protein or peptide: C8 beta
  • Protein or peptide: C8 gamma
  • Protein or peptide: C9

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Supramolecule #1000: Membrane attack complex

SupramoleculeName: Membrane attack complex / type: sample / ID: 1000
Details: Protein complex was assembled on liposomes and detergent solubilized
Number unique components: 7
Molecular weightTheoretical: 1.8 MDa

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Macromolecule #1: C5

MacromoleculeName: C5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 190 KDa

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Macromolecule #2: C6

MacromoleculeName: C6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 120 KDa

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Macromolecule #3: C7

MacromoleculeName: C7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 110 KDa

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Macromolecule #4: C8 alpha

MacromoleculeName: C8 alpha / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 152 KDa

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Macromolecule #5: C8 beta

MacromoleculeName: C8 beta / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 152 KDa

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Macromolecule #6: C8 gamma

MacromoleculeName: C8 gamma / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 152 KDa

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Macromolecule #7: C9

MacromoleculeName: C9 / type: protein_or_peptide / ID: 7 / Number of copies: 18 / Oligomeric state: eighteen-mer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Plasma
Molecular weightTheoretical: 69 KDa

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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 / Details: 20 mM HEPES, 150 mM NaCl
GridDetails: 300 mesh quatifol R1.2/1.3 grids with thin carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.00 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJul 2, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14.0 µm / Number real images: 622 / Average electron dose: 45 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3, phase flip on each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: OTHER / Software - Name: RELION, EMAN2 / Number images used: 25343
DetailsMasked refinement

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