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- PDB-9fmx: Aerolysin Y221G - prepore -

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Basic information

Entry
Database: PDB / ID: 9fmx
TitleAerolysin Y221G - prepore
ComponentsAerolysin
KeywordsTOXIN / pore forming toxin / aerolysin / cryo-EM
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: 2.2 Å
AuthorsIacovache, I. / Zuber, B.
Funding support Switzerland, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179520 Switzerland
Swiss National Science Foundation32NE30_185536 Switzerland
Swiss National Science Foundation32NE30_185536 Switzerland
Citation
Journal: J Am Chem Soc / Year: 2025
Title: Aerolysin Nanopore Structures Revealed at High Resolution in a Lipid Environment.
Authors: Jana S Anton / Ioan Iacovache / Juan F Bada Juarez / Luciano A Abriata / Louis W Perrin / Chan Cao / Maria J Marcaida / Benoît Zuber / Matteo Dal Peraro /
Abstract: Aerolysin is a β-pore-forming toxin produced by most Aeromonas bacteria, which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally ...Aerolysin is a β-pore-forming toxin produced by most Aeromonas bacteria, which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging to the aerolysin-like family, are present throughout all kingdoms of life, but very few of them have been structurally characterized in a lipid environment. Here, we present the first high-resolution atomic cryo-EM structures of aerolysin prepore and pore in a membrane-like environment. These structures allow the identification of key interactions, which are relevant for understanding the pore formation mechanism and for correctly positioning the pore β-barrel and its anchoring β-turn motif in the membrane. Moreover, we elucidate at high resolution the architecture of key pore mutations and precisely identify four constriction rings in the pore lumen that are highly relevant for nanopore sensing experiments.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionJun 7, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)741,23714
Polymers741,23714
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Aerolysin


Mass: 52945.473 Da / Num. of mol.: 14 / Mutation: Y221G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas hydrophila (bacteria) / Gene: aerA / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / References: UniProt: P09167
Has protein modificationY

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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 pre-pore Y221G double heptamer / Type: COMPLEX
Details: Heptamerization was induced by trypsinization of proaerolysin Y221G.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Aeromonas hydrophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: DE3 / Plasmid: pet22c
Buffer solutionpH: 7.4 / Details: 20mM Hepes 7.4 100mM NaCl 0.05% A8-35
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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 / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF ChimeraXmodel fitting
8UCSF Chimeramodel fitting
9Cootmodel fitting
11PHENIX1.21_5207model refinement
13RELION5final Euler assignment
14RELION5classification
15RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Details: ModelAngelo was used with the final map to generate the first model.
Atomic model buildingDetails: modelangelo / Source name: Other / Type: other
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00547894
ELECTRON MICROSCOPYf_angle_d0.47165394
ELECTRON MICROSCOPYf_dihedral_angle_d9.72517024
ELECTRON MICROSCOPYf_chiral_restr0.0496832
ELECTRON MICROSCOPYf_plane_restr0.0048554

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