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- PDB-2bk1: The pore structure of pneumolysin, obtained by fitting the alpha ... -

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Entry
Database: PDB / ID: 2bk1
TitleThe pore structure of pneumolysin, obtained by fitting the alpha carbon trace of perfringolysin O into a cryo-EM map
ComponentsPERFRINGOLYSIN O
KeywordsTOXIN / CYTOLYSIS / HEMOLYSIS / THIOL-ACTIVATED CYTOLYSIN / CRYOEM / CYTOLYTIC PROTEIN
Function / homology
Function and homology information


hemolysis in another organism / cholesterol binding / toxin activity / membrane => GO:0016020 / 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
Perfringolysin O / Perfringolysin O
Similarity search - Component
Biological speciesCLOSTRIDIUM PERFRINGENS (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 29 Å
Model type detailsCA ATOMS ONLY, CHAIN A
AuthorsTilley, S.J. / Orlova, E.V. / Gilbert, R.J.C. / Andrew, P.W. / Saibil, H.R.
Citation
Journal: 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.
#1: Journal: Cell / Year: 1997
Title: Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form.
Authors: J Rossjohn / S C Feil / W J McKinstry / R K Tweten / M W Parker /
Abstract: The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member ...The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.
History
DepositionFeb 10, 2005Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 4, 2005Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2013Group: Version format compliance
Revision 1.2Apr 19, 2017Group: Other

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Assembly

Deposited unit
A: PERFRINGOLYSIN O


Theoretical massNumber of molelcules
Total (without water)50,0961
Polymers50,0961
Non-polymers00
Water0
1
A: PERFRINGOLYSIN O
x 38


Theoretical massNumber of molelcules
Total (without water)1,903,64438
Polymers1,903,64438
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation38
2


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C38 (38 fold cyclic))

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Components

#1: Protein PERFRINGOLYSIN O / THETA-TOXIN / THIOL-ACTIVATED CYTOLYSIN / Coordinate model: Cα atoms only


Mass: 50095.902 Da / Num. of mol.: 1 / Fragment: RESIDUES 53-500
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM PERFRINGENS (bacteria) / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P19995, UniProt: P0C2E9*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PNEUMOLYSIN / Type: COMPLEX
Details: THE SAMPLE CONSISTS OF PNEUMOLYSIN IN A MEMBRANE-INSERTED PORE STATE
Buffer solutionName: 8 MM NA2HP04, 1.5MM KH2PO4, 2.5 MM KCL, 0.25 MM NACL / pH: 6.95
Details: 8 MM NA2HP04, 1.5MM KH2PO4, 2.5 MM KCL, 0.25 MM NACL
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: PLUNGED INTO ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Details: SAMPLES WERE MAINTAINED AT LIQUID NITROGEN TEMPERATURES IN THE ELECTRON MICROSCOPE.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 42000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1100 nm / Cs: 2 mm
Specimen holderTemperature: 100 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 135
Radiation wavelengthRelative weight: 1

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Processing

CTF correctionDetails: PHASE FLIPPING
SymmetryPoint symmetry: C38 (38 fold cyclic)
3D reconstructionResolution: 29 Å / Num. of particles: 88 / Nominal pixel size: 3.5 Å
Details: RESIDUES 134-142 ARE MISSING FROM THE SEQUENCE. RESIDUES CORRESPONDING TO TO TM1 AND TM2 (187-220,284-315) WERE MODELLED AS A POLY-ALANINE FLAT BETA HAIRPINS. THE OLIGOMER CAN BE GENERATED ...Details: RESIDUES 134-142 ARE MISSING FROM THE SEQUENCE. RESIDUES CORRESPONDING TO TO TM1 AND TM2 (187-220,284-315) WERE MODELLED AS A POLY-ALANINE FLAT BETA HAIRPINS. THE OLIGOMER CAN BE GENERATED BY APPLYING 38-FOLD ROTATIONAL SYMMETRY.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: METHOD--THE CRYSTAL STRUCTURE OF PERFRINOGLYSIN O (1PFO, ROSSJOHN ET AL., 1998, CELL 89, 685)WAS PLACED INTO THE CRYO-EM DENSITY MAP (EMD-1107). THE ALPHA CARBON TRACE OF PERFRINGOLYSIN 0 ...Details: METHOD--THE CRYSTAL STRUCTURE OF PERFRINOGLYSIN O (1PFO, ROSSJOHN ET AL., 1998, CELL 89, 685)WAS PLACED INTO THE CRYO-EM DENSITY MAP (EMD-1107). THE ALPHA CARBON TRACE OF PERFRINGOLYSIN 0 WAS MANUALLY POSITIONED INTO THE CRYO-EM DENSITY CORRESPONDING TO THE POSITION OF ONE SUBUNIT. THE BEST FIT WAS OBTAINED BY SEPARATING THE MONOMER 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). THE COMPLETE OLIGOMER (38-MER) WAS GENERATED AND CHECKED FOR CLOSE CONTACTS BOTH BY EYE AND USING THE CCP4 PROGRAM CONTACT.TO IMPROVE THE FIT SECTIONS CORRESPONDING TO 3 SUBUNITS WERE EXTRACTED FROM THE 38-MER MAP (EMD-1107) AND A 44-MER MAP AND ALIGNED. THE WEIGHTED AVERAGE WAS CALCULATED AND THE IMPROVED MAP USED FOR THE FINAL MANUAL FITTING. THE TWO SECTIONS ARE CONSISTENT TO A RESOLUTION OF 28 ANGSTROMS (0.5 CORRELATION FSC) AND THE WEIGHTED AVERAGE MAP WAS RECONSTRUCTED FROM 131 PARTICLES.
Atomic model buildingPDB-ID: 1PFO

1pfo

RefinementHighest resolution: 29 Å
Refinement stepCycle: LAST / Highest resolution: 29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms444 0 0 0 444

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