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- EMDB-1107: Structural basis of pore formation by the bacterial toxin pneumolysin. -

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Basic information

Entry
Database: EMDB / ID: EMD-1107
TitleStructural basis of pore formation by the bacterial toxin pneumolysin.
Map dataThis is the 3D map for the pore form of pneumolysin, calculated using c38 symmetry.
Sample
  • Sample: Pneumolysin
  • Organelle or cellular component: Phosphatidylcholine-cholesterol lipid bilayer
  • Protein or peptide: Pneumolysin
Function / homology
Function and homology information


hemolysis in another organism / cholesterol binding / membrane => GO:0016020 / toxin activity / 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 / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin
Similarity search - Domain/homology
Perfringolysin O / Perfringolysin O
Similarity search - Component
Biological speciessynthetic construct (others) / Streptococcus pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 29.0 Å
AuthorsTilley SJ / Orlova EV / Gilbert RJ / Andrew PW / Saibil HR
CitationJournal: Cell / Year: 2005
Title: Structural basis of pore formation by the bacterial toxin pneumolysin.
Authors: Sarah J Tilley / Elena V Orlova / Robert J C Gilbert / Peter W Andrew / Helen R Saibil /
Abstract: The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM ...The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.
History
DepositionJan 19, 2005-
Header (metadata) releaseJan 20, 2005-
Map releaseJan 20, 2005-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2bk1
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2bk1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1107.gif

Downloads & links

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Map

FileDownload / File: emd_1107.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D map for the pore form of pneumolysin, calculated using c38 symmetry.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
3.5 Å/pix.
x 160 pix.
= 560. Å		3.5 Å/pix.
x 160 pix.
= 560. Å		3.5 Å/pix.
x 160 pix.
= 560. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.5 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.00697 / Movie #1: 0.04
Minimum - Maximum-0.170322 - 0.21269
Average (Standard dev.)-0.0000256958 (±0.0235138)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 560 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z560.000560.000560.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-80-80-80
NX/NY/NZ160160160
MAP C/R/S213
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.1700.213-0.000

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Supplemental data

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Sample components

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Entire : Pneumolysin

EntireName: Pneumolysin
Components
  • Sample: Pneumolysin
  • Organelle or cellular component: Phosphatidylcholine-cholesterol lipid bilayer
  • Protein or peptide: Pneumolysin

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Supramolecule #1000: Pneumolysin

SupramoleculeName: Pneumolysin / type: sample / ID: 1000 / Oligomeric state: 38-mer / Number unique components: 2
Molecular weightTheoretical: 2.0 MDa

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Supramolecule #1: Phosphatidylcholine-cholesterol lipid bilayer

SupramoleculeName: Phosphatidylcholine-cholesterol lipid bilayer / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No
Source (natural)Organism: synthetic construct (others) / synonym: membrane

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Macromolecule #1: Pneumolysin

MacromoleculeName: Pneumolysin / type: protein_or_peptide / ID: 1 / Number of copies: 38 / Oligomeric state: 38-mer / Recombinant expression: Yes
Source (natural)Organism: Streptococcus pneumoniae (bacteria) / Location in cell: cytoplasm
Molecular weightExperimental: 2.0 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pKK233-2
SequenceInterPro: Thiol-activated cytolysin

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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

Concentration0.05 mg/mL
BufferpH: 6.95
Details: 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.5 mM KCl, 0.25M NaCl
GridDetails: holey carbon 400 mesh copper grid, glow discharged using positive charge
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home made plunger
Method: Grids were blotted for approximately 3 seconds and allowed to drain vertically for 5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 100 K / Max: 100 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: corrected at 150,000 magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 135 / Average electron dose: 20 e/Å2 / Details: After scanning images were averaged 2x2. / Od range: 1 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Side entry / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsPrior to vitrification pneumolysin was added to freshly prepared liposomes consisting of 10:10:1 molar ratio of PC: cholestrol: dicetylphosphate. Pneumolysin was added at a molar ratio of 1:2000 for dialysed liposomes or 1:4000 for extruded liposomes and placed on ice for 5 minutes. This mixture was incubated at 37C for 3 minutes before application to the grid.
CTF correctionDetails: phase flipping
Final reconstructionApplied symmetry - Point group: C38 (38 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic
Details: Weighted back projection and amplitude scaling were used.
Number images used: 88
Final angle assignmentDetails: Tomographic series around symmetry axis
Final two d classificationNumber classes: 11

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Atomic model buiding 1

Initial modelPDB ID:

1pfo

DetailsThe 1pfo structure was separated into six rigid bodies: domain 1 (91-172, 231-272, 354-373), domain 2 upper (53-62, 83-90, 374-381), domain 2 lower (63-82, 382-390), domain 3 (177-186, 221-230, 273-283, 316-353), domain 3 hairpins (187-220, 284-315), and domain 4 (391-500). These rigid bodies were fitted manually using the software O and pymol.
RefinementProtocol: RIGID BODY FIT
Output model

PDB-2bk1:
The pore structure of pneumolysin, obtained by fitting the alpha carbon trace of perfringolysin O into a cryo-EM map

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