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- PDB-5jzw: Cryo-EM structures of aerolysin post-prepore and quasipore -

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Basic information

Entry
Database: PDB / ID: 5jzw
TitleCryo-EM structures of aerolysin post-prepore and quasipore
ComponentsAerolysin
KeywordsTOXIN / pore forming toxin / concentric beta-barrel / aerolysin
Function / homology
Function and homology information


toxin activity / host cell plasma membrane / extracellular region / identical protein binding / membrane
Similarity search - Function
Aerolysin / Aerolysin toxin / Aerolysin toxin / Aerolysin/Pertussis toxin domain / Aerolysin/Pertussis toxin (APT), N-terminal domain superfamily / Aerolysin/Pertussis toxin (APT) domain / Aerolysin/haemolysin toxin, conserved site / Aerolysin type toxins signature. / C-type lectin fold
Similarity search - Domain/homology
Biological speciesAeromonas hydrophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.46 Å
AuthorsIacovache, I. / Zuber, B.
CitationJournal: Nat Commun / Year: 2016
Title: Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process.
Authors: Ioan Iacovache / Sacha De Carlo / Nuria Cirauqui / Matteo Dal Peraro / F Gisou van der Goot / Benoît Zuber /
Abstract: Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural ...Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.
History
DepositionMay 17, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jul 13, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name
Revision 1.2Nov 21, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type

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Structure visualization

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Assembly

Deposited unit
A: Aerolysin
B: Aerolysin
C: Aerolysin
D: Aerolysin
E: Aerolysin
F: Aerolysin
G: Aerolysin
H: Aerolysin
I: Aerolysin
J: Aerolysin
K: Aerolysin
L: Aerolysin
M: Aerolysin
N: Aerolysin


Theoretical massNumber of molelcules
Total (without water)659,86414
Polymers659,86414
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area74320 Å2
ΔGint-53 kcal/mol
Surface area247960 Å2
MethodPISA

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Components

#1: Protein
Aerolysin


Mass: 47133.145 Da / Num. of mol.: 14 / Mutation: K246C, E258C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas hydrophila (bacteria) / Gene: aerA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P09167

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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: aerolysin post-prepore and quasipore / Type: COMPLEX
Details: Aerolysin oligomer arrested in post-prepore and quasipore states by the K246C/E258C mutation.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Aeromonas hydrophila (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: PET22b
Buffer solutionpH: 7.4
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 295 K
Details: 30 seconds glow discharged grids, 4ul sample, 5 seconds blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 1 sec. / Electron dose: 22.42 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.3particle selection
2EPUimage acquisitionimage acquisition
3RELIONimage acquisitionimage processing
5RELION1.3CTF correction
8Rosettamodel fitting
11RELION1.3initial Euler assignment
14RELION1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 260958
3D reconstructionResolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70766 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 3C0N

3c0n


Pdb chain-ID: B

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