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- EMDB-3289: Subtomogram average of the membrane attack complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3289
TitleSubtomogram average of the membrane attack complex
Map dataSubtomogram average of the complement membrane attack complex
Sample
  • Sample: Subtomogram average of the membrane attack complex pore in a lipid bilayer
  • Protein or peptide: Complement component C5b6
  • Protein or peptide: Complement component C7
  • Protein or peptide: Complement component C8-alpha chain
  • Protein or peptide: Complement component C8-beta chain
  • Protein or peptide: Complement component C8-gamma chain
  • Protein or peptide: Complement component C9 chain
Keywordsvolta phase plate / membrane attack complex / complement / electron tomography / sub-tomogram average / pore
Function / homology
Function and homology information


cell killing / Terminal pathway of complement / membrane attack complex / complement binding / other organism cell membrane / complement activation / complement activation, alternative pathway / retinol binding / complement activation, classical pathway / Regulation of Complement cascade ...cell killing / Terminal pathway of complement / membrane attack complex / complement binding / other organism cell membrane / complement activation / complement activation, alternative pathway / retinol binding / complement activation, classical pathway / Regulation of Complement cascade / protein homooligomerization / positive regulation of immune response / extracellular vesicle / killing of cells of another organism / blood microparticle / immune response / protein-containing complex binding / extracellular space / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
Kazal-type serine protease inhibitor domain / : / : / Kazal-type serine protease inhibitor domain / Complement component C7, FIM2 N-terminal / Complement component C7, Kazal domain / Complement component C8 gamma chain / : / Complement components C8A/B/C6, EGF-like domain / Membrane attack complex component/perforin/complement C9 ...Kazal-type serine protease inhibitor domain / : / : / Kazal-type serine protease inhibitor domain / Complement component C7, FIM2 N-terminal / Complement component C7, Kazal domain / Complement component C8 gamma chain / : / Complement components C8A/B/C6, EGF-like domain / Membrane attack complex component/perforin/complement C9 / Alpha-1-microglobulin / Factor I / membrane attack complex / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / factor I membrane attack complex / membrane-attack complex / perforin / MAC/Perforin domain / Membrane attack complex/perforin (MACPF) domain profile. / Membrane attack complex component/perforin (MACPF) domain / Thrombospondin type 1 domain / 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 / Lipocalin family conserved site / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Lipocalin / cytosolic fatty-acid binding protein family / Lipocalin/cytosolic fatty-acid binding domain / Calycin / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 1. / EGF-like domain signature 2. / Lipocalin signature.
Similarity search - Domain/homology
Complement component C9 / Complement component C8 alpha chain / Complement component C8 beta chain / Complement component C8 gamma chain / Complement component C7
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 23.0 Å
AuthorsSharp TH / Koster AJ / Gros P
CitationJournal: Cell Rep / Year: 2016
Title: Heterogeneous MAC Initiator and Pore Structures in a Lipid Bilayer by Phase-Plate Cryo-electron Tomography.
Authors: Thomas H Sharp / Abraham J Koster / Piet Gros /
Abstract: Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding ...Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding and membrane insertion of C5b6, C7, C8, and C9. Using cryo-electron tomography with a Volta phase plate and subtomogram averaging, we imaged C5b-7, C5b-8, and C5b-9 complexes and determined the C5b-9 pore structure in lipid bilayers. The in situ C5b-9 pore structure at 2.3-nm resolution reveals a 10- to 11.5-nm cone-shaped pore starting with C5b678 and multiple copies of C9 that is poorly closed, yielding a seam between C9 and C6 substituting for the shorter β strands in C6 and C7. However, large variations of composite pore complexes are apparent in subtomograms. Oligomerized initiator complexes C5b-7 and C5b-8 show stages of membrane binding, deformation, and perforation that yield ∼3.5-nm-wide pores. These data indicate a dynamic process of pore formation that likely adapts to biological membranes under attack.
History
DepositionDec 27, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseMar 30, 2016-
UpdateMar 30, 2016-
Current statusMar 30, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.11
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3289.png

Downloads & links

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Map

FileDownload / File: emd_3289.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the complement membrane attack complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.86 Å/pix.
x 240 pix.
= 686.64 Å		2.86 Å/pix.
x 240 pix.
= 686.64 Å		2.86 Å/pix.
x 240 pix.
= 686.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.861 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11 / Movie #1: 0.11
Minimum - Maximum-0.1858658 - 0.41718206
Average (Standard dev.)0.00101197 (±0.0297654)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 686.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.8612.8612.861
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z686.640686.640686.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.1860.4170.001

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Supplemental data

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Sample components

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Entire : Subtomogram average of the membrane attack complex pore in a lipi...

EntireName: Subtomogram average of the membrane attack complex pore in a lipid bilayer
Components
  • Sample: Subtomogram average of the membrane attack complex pore in a lipid bilayer
  • Protein or peptide: Complement component C5b6
  • Protein or peptide: Complement component C7
  • Protein or peptide: Complement component C8-alpha chain
  • Protein or peptide: Complement component C8-beta chain
  • Protein or peptide: Complement component C8-gamma chain
  • Protein or peptide: Complement component C9 chain

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Supramolecule #1000: Subtomogram average of the membrane attack complex pore in a lipi...

SupramoleculeName: Subtomogram average of the membrane attack complex pore in a lipid bilayer
type: sample / ID: 1000
Oligomeric state: One copy of C5b, C6 and C7; one C8 heterotrimer; multiple copies of C9
Number unique components: 6

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Macromolecule #1: Complement component C5b6

MacromoleculeName: Complement component C5b6 / type: protein_or_peptide / ID: 1 / Name.synonym: C5b6 / Number of copies: 1 / Oligomeric state: Heterodimer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 285 KDa

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Macromolecule #2: Complement component C7

MacromoleculeName: Complement component C7 / type: protein_or_peptide / ID: 2 / Name.synonym: C7 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 92.4 KDa
SequenceUniProtKB: Complement component C7

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Macromolecule #3: Complement component C8-alpha chain

MacromoleculeName: Complement component C8-alpha chain / type: protein_or_peptide / ID: 3 / Name.synonym: C8alpha / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 64 KDa
SequenceUniProtKB: Complement component C8 alpha chain

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Macromolecule #4: Complement component C8-beta chain

MacromoleculeName: Complement component C8-beta chain / type: protein_or_peptide / ID: 4 / Name.synonym: C8beta / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 64 KDa
SequenceUniProtKB: Complement component C8 beta chain

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Macromolecule #5: Complement component C8-gamma chain

MacromoleculeName: Complement component C8-gamma chain / type: protein_or_peptide / ID: 5 / Name.synonym: C8gamma / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 22 KDa
SequenceUniProtKB: Complement component C8 gamma chain

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Macromolecule #6: Complement component C9 chain

MacromoleculeName: Complement component C9 chain / type: protein_or_peptide / ID: 6 / Name.synonym: C9 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Serum
Molecular weightTheoretical: 71 KDa
SequenceUniProtKB: Complement component C9

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.241 mg/mL
BufferpH: 7.9 / Details: 10 mM Tris-HCl, 30 mM NaCl
GridDetails: 300 mesh copper grids with lacey-carbon support, glow discharged.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90.15 K / Instrument: LEICA EM GP
Details: 6 ul sample pipetted onto freshly plasma-cleaned 300 mesh copper grids with lacey-carbon support
Method: Blot for 6 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsLow-dose protocol used. Volta phase plate used
DateMar 10, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 1 e/Å2 / Details: Volta phase plate used
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: 60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsSubtomograms were picked by hand
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN2
Details: Initial model was phase-randomized beyond 6 nm. Refinement was against a rotationally-averaged map.
Number subtomograms used: 986
Final 3D classificationNumber classes: 1
FSC plot (resolution estimation)

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