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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/homologyThiol-activated cytolysins signature. / Thiol-activated cytolysin / Thiol-activated cytolysin C-terminal / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin / Thiol-activated cytolysin beta sandwich domain / cholesterol binding / hemolysis in other organism / toxin activity ...Thiol-activated cytolysins signature. / Thiol-activated cytolysin / Thiol-activated cytolysin C-terminal / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin / Thiol-activated cytolysin beta sandwich domain / cholesterol binding / hemolysis in other organism / toxin activity / host cell plasma membrane / integral component of membrane / extracellular region / Pneumolysin
Function and homology information
Specimen sourceStreptococcus pneumoniae serotype 2 / / bacteria
MethodElectron microscopy (4.5 Å resolution / Particle / Single particle) / Transmission electron microscopy
Authorsvan Pee, K. / Neuhaus, A. / D'Imprima, E. / Mills, D.J. / Kuehlbrandt, W. / Yildiz, O.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 24, 2016 / Release: Apr 5, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 5, 2017Structure modelrepositoryInitial release
1.1Aug 2, 2017Structure modelData collectionem_image_scans / em_software_em_software.name

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Assembly

Deposited unit
B: Pneumolysin


Theoretical massNumber of molelcules
Total (without water)52,8661
Polyers52,8661
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)0
ΔGint (kcal/M)0
Surface area (Å2)29420
MethodPISA

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Components

#1: Protein/peptide Pneumolysin / / Thiol-activated cytolysin


Mass: 52866.066 Da / Num. of mol.: 1
Source: (gene. exp.) Streptococcus pneumoniae serotype 2 (strain d39 / nctc 7466) / / bacteria
Gene: ply, SPD_1726 / Production host: Escherichia coli BL21(DE3) / References: UniProt:Q04IN8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Pneumolysin pore complex / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 2.2 deg. / Units: MEGADALTONS
Source (natural)Organism: Streptococcus pneumoniae
Source (recombinant)Organism: Escherichia coli BL21 / 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
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
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
12RELION1.4RECONSTRUCTION
13Coot8.4MODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C42
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 6461 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 5AOD
Pdb chain ID: A / Pdb chain residue range: 1-471

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