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

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Basic information

Entry
Database: PDB / ID: 9eyo
TitleThe structure of solubilized octameric pore of actinoporin Fav prepared on POPG, 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: 2.86 Å
AuthorsSolinc, G. / Srnko, M. / Anderluh, G. / Podobnik, M.
Funding support Slovenia, 3items
OrganizationGrant numberCountry
Slovenian Research AgencyJ4-8225 Slovenia
Slovenian Research AgencyP1-0391 Slovenia
Other private
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM structures of a protein pore reveal a cluster of cholesterol molecules and diverse roles of membrane lipids.
Authors: Gašper Šolinc / Marija Srnko / Franci Merzel / Ana Crnković / Mirijam Kozorog / Marjetka Podobnik / Gregor Anderluh /
Abstract: The structure and function of membrane proteins depend on their interactions with lipids that constitute membranes. Actinoporins are α-pore-forming proteins that bind preferentially to sphingomyelin- ...The structure and function of membrane proteins depend on their interactions with lipids that constitute membranes. Actinoporins are α-pore-forming proteins that bind preferentially to sphingomyelin-containing membranes, where they oligomerize and form transmembrane pores. Through a comprehensive cryo-electron microscopic analysis of a pore formed by an actinoporin Fav from the coral Orbicella faveolata, we show that the octameric pore interacts with 112 lipids in the upper leaflet of the membrane, reveal the roles of lipids, and demonstrate that the actinoporin surface is suited for binding multiple receptor sphingomyelin molecules. When cholesterol is present in the membrane, it forms a cluster of four molecules associated with each protomer. Atomistic simulations support the structural data and reveal additional effects of the pore on the lipid membrane. These data reveal a complex network of protein-lipid and lipid-lipid interactions and an underrated role of lipids in the structure and function of transmembrane protein complexes.
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
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Revision 1.0Apr 23, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Nov 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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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)260,817128
Polymers184,0208
Non-polymers76,797120
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
Actinoporin / Fav


Mass: 23002.561 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: This 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) / 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.: 88 / 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: Details: Octameric Fav pore prepared on 1-Palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG):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.: 2 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: 2600 nm / Nominal defocus min: 600 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) / Num. of real images: 11470
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: 2705265 / Details: Number of particles after template picking
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118851 / 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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