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- PDB-9eyn: The structure of solubilized octameric pore of actinoporin Fav pr... -

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Basic information

Entry
Database: PDB / ID: 9eyn
TitleThe structure of solubilized octameric pore of actinoporin Fav prepared on DOPC, cholesterol, sphingomyelin membranes
ComponentsActinoporin
KeywordsTOXIN / Actinoporin / Pore-forming toxin / Pore / Octamer / Transmembrane pore / cholesterol / Nanopore
Function / homology: / CHOLESTEROL
Function and homology information
Biological speciesOrbicella faveolata (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsSolinc, G. / Srnko, M. / Svigel, T. / Anderluh, G. / Podobnik, M.
Funding support Slovenia, 3items
OrganizationGrant numberCountry
Slovenian Research AgencyJ4-8225 Slovenia
Slovenian Research AgencyP1-0391 Slovenia
Other private
CitationJournal: To Be Published
Title: The structure of solubilized octameric pore of actinoporin Fav prepared on DOPC:sphingomyelin membranes
Authors: Solinc, G. / Srnko, M. / Anderluh, G. / Podobnik, M.
History
DepositionApr 9, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actinoporin
B: Actinoporin
C: Actinoporin
D: Actinoporin
E: Actinoporin
F: Actinoporin
G: Actinoporin
H: Actinoporin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)295,509112
Polymers230,4268
Non-polymers65,083104
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53320 Å2
ΔGint111 kcal/mol
Surface area61190 Å2
MethodPISA

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Components

#1: Protein
Actinoporin / Fav


Mass: 28803.260 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: The protein was expressed with an N-terminal addition of 6xHis tag and TEV cleavage site. After removing the His-tag, the final construct has three additional residues at the N-terminal (GHM).
Source: (gene. exp.) Orbicella faveolata (invertebrata) / Plasmid: pET28a (+)
Details (production host): modified pET28a (+) plasmid with the N-terminal 6 histidine tag and TEV restriction site (ENLYFQGHM)
Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical...
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 32 / Source method: obtained synthetically / Formula: C27H46O
#3: Chemical...
ChemComp-A1H8M / Sphingomyelin C18 / trimethyl-[2-[[(E,2S,3R)-2-(octadecanoylamino)-3-oxidanyl-octadec-4-enoxy]-oxidanyl-phosphoryl]oxyethyl]azanium


Mass: 732.089 Da / Num. of mol.: 72 / Source method: obtained synthetically / Formula: C41H84N2O6P
Has ligand of interestN
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: Octameric Fav pore in complex with sphingomyelin and cholesterol molecules solubilized by detergents
Type: COMPLEX
Details: Octameric Fav pore prepared on 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC):sphingomyelin:cholesterol (1:1:1 molar ratio) membranes
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Orbicella faveolata (invertebrata)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Cell: DE3 / Plasmid: pET28a
Buffer solutionpH: 8 / Details: 150 mM NaCl, 50 mM Tris/HCl, 0.02 % Brij 35, pH 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
250 mMTris1
30.02 %Brij 351
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4particle selection
2EPUimage acquisition
4cryoSPARCv4CTF correction
7UCSF ChimeraX1.6model fitting
9cryoSPARCV4initial Euler assignment
10cryoSPARCv4final Euler assignment
11cryoSPARCv4classification
12cryoSPARCv43D reconstruction
13Coot0.98model refinement
14PHENIX1.19model refinement
15ISOLDE1.6model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1266343 / Details: Number of particles after template picking
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 223527 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingDetails: Pore structure of the same protein from related entries
Source name: Other / Type: experimental model

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