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- EMDB-2612: Incomplete pneumolysin oligomers form membrane pores -

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Basic information

Entry
Database: EMDB / ID: EMD-2612
TitleIncomplete pneumolysin oligomers form membrane pores
Map dataSub-tomogram average of 197 pre-pores.
Sample
  • Sample: Pneumolysin prepore formed on a cholesterol-containing liposome
  • Organelle or cellular component: synthetic membrane
  • Protein or peptide: pneumolysin
Keywordscholesterol-dependent cytolysin / pneumolysin / proteolipid toroidal pore
Function / homology
Function and homology information


cholesterol binding / toxin activity / killing of cells of another organism / host cell plasma membrane / extracellular region / membrane
Similarity search - Function
Thiol-activated cytolysins signature. / Thiol-activated cytolysin C-terminal / Thiol-activated cytolysin, C-terminal domain superfamily / Thiol-activated cytolysin beta sandwich domain / Thiol-activated cytolysin / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin
Similarity search - Domain/homology
Biological speciessynthetic construct (others) / Streptococcus pneumoniae (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 29.0 Å
AuthorsSonnen AF-P / Plitzko JM / Gilbert RJC
CitationJournal: Open Biol / Year: 2014
Title: Incomplete pneumolysin oligomers form membrane pores.
Authors: Andreas F-P Sonnen / Jürgen M Plitzko / Robert J C Gilbert /
Abstract: Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize ...Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.
History
DepositionMar 25, 2014-
Header (metadata) releaseApr 9, 2014-
Map releaseMay 7, 2014-
UpdateMay 7, 2014-
Current statusMay 7, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 10.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD2612.gif

Downloads & links

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Map

FileDownload / File: emd_2612.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram average of 197 pre-pores.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.72 Å/pix.
x 104 pix.
= 490.88 Å		4.72 Å/pix.
x 104 pix.
= 490.88 Å		4.72 Å/pix.
x 104 pix.
= 490.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 10.699999999999999 / Movie #1: 10.7
Minimum - Maximum-47.705104830000003 - 66.461158749999996
Average (Standard dev.)1.89449048 (±8.68987179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions104104104
Spacing104104104
CellA=B=C: 490.87997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.724.724.72
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z490.880490.880490.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS104104104
D min/max/mean-47.70566.4611.894

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Supplemental data

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Sample components

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Entire : Pneumolysin prepore formed on a cholesterol-containing liposome

EntireName: Pneumolysin prepore formed on a cholesterol-containing liposome
Components
  • Sample: Pneumolysin prepore formed on a cholesterol-containing liposome
  • Organelle or cellular component: synthetic membrane
  • Protein or peptide: pneumolysin

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Supramolecule #1000: Pneumolysin prepore formed on a cholesterol-containing liposome

SupramoleculeName: Pneumolysin prepore formed on a cholesterol-containing liposome
type: sample / ID: 1000 / Oligomeric state: Full ring oligomer / Number unique components: 2
Molecular weightTheoretical: 2 MDa

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Supramolecule #1: synthetic membrane

SupramoleculeName: synthetic membrane / type: organelle_or_cellular_component / ID: 1
Details: Liposome formed of a 10:10:1 mixture of phosphatidylcholine:cholesterol:dicetyl phosphate
Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: pneumolysin

MacromoleculeName: pneumolysin / type: protein_or_peptide / ID: 1
Details: pneumolysin added to the synthetic membranes spontaneously forms oligomers on their surfaces.
Oligomeric state: 40mer / Recombinant expression: Yes
Source (natural)Organism: Streptococcus pneumoniae (bacteria) / Location in cell: cytoplasm
Molecular weightExperimental: 52 KDa / Theoretical: 52 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: M15 / Recombinant plasmid: pKK233-2
SequenceUniProtKB: Pneumolysin

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: PBS
GridDetails: C-flat or lacy carbon-coated grids
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: OTHER

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateJun 1, 2008
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 64171
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsSubtomograms were selected manually in Bshow and subjected to automatic maximum-likelihood based classification using XMIPP.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: OTHER / Software - Name: TOM, Toolbox, Bsoft, XMIPP / Number subtomograms used: 197

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