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- PDB-5ly6: CryoEM structure of the membrane pore complex of Pneumolysin at 4.5A -

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Basic information

Entry
Database: PDB / ID: 5ly6
TitleCryoEM structure of the membrane pore complex of Pneumolysin at 4.5A
ComponentsPneumolysin
KeywordsTOXIN / Bacterial toxins / pore complex / cryoEM structure / cholesterol-dependent cytolysin / Pneumolysin / membrane 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 speciesStreptococcus pneumoniae serotype 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
Authorsvan Pee, K. / Neuhaus, A. / D'Imprima, E. / Mills, D.J. / Kuehlbrandt, W. / Yildiz, O.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck SocietyBiophysics/Structural Biology Germany
CitationJournal: Elife / Year: 2017
Title: CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.
Authors: Katharina van Pee / Alexander Neuhaus / Edoardo D'Imprima / Deryck J Mills / Werner Kühlbrandt / Özkan Yildiz /
Abstract: Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of ...Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of , by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.
History
DepositionSep 24, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name
Revision 1.2Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: em_admin / pdbx_data_processing_status ...em_admin / pdbx_data_processing_status / pdbx_database_proc / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _em_admin.last_update
Revision 1.3Oct 2, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.length_a / _cell.length_b / _cell.length_c

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Structure visualization

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Assembly

Deposited unit
B: Pneumolysin


Theoretical massNumber of molelcules
Total (without water)52,8661
Polymers52,8661
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area29420 Å2
MethodPISA

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Components

#1: Protein Pneumolysin / / Thiol-activated cytolysin


Mass: 52866.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae serotype 2 (strain D39 / NCTC 7466) (bacteria)
Gene: ply, SPD_1726 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q04IN8

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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 pore complex / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 2.2 MDa
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Plasmid: pET15
Buffer solutionpH: 7
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 6 sec. / Electron dose: 1.02 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION1.4particle selection
2RELION1.4image acquisition
4CTFFIND3CTF correction
7Coot8.4model fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13Coot8.4model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C42 (42 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6461 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 5AOD

5aod


Pdb chain-ID: A / Pdb chain residue range: 1-471

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