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- EMDB-50058: The structure of solubilized octameric pore of actinoporin Fav pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-50058
TitleThe structure of solubilized octameric pore of actinoporin Fav prepared on DOPC, cholesterol, sphingomyelin membranes
Map dataMain map
Sample
  • Complex: Octameric Fav pore in complex with sphingomyelin and cholesterol molecules solubilized by detergents
    • Protein or peptide: Actinoporin
  • Ligand: CHOLESTEROL
  • Ligand: Sphingomyelin C18
KeywordsActinoporin / Pore-forming toxin / Pore / Octamer / Transmembrane pore / TOXIN / cholesterol / Nanopore
Biological speciesOrbicella faveolata (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsSolinc G / Srnko M / Svigel T / Anderluh G / Podobnik M
Funding support Slovenia, 3 items
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, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50058.png

Downloads & links

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Map

FileDownload / File: emd_50058.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 320 pix.
= 302.624 Å		0.95 Å/pix.
x 320 pix.
= 302.624 Å		0.95 Å/pix.
x 320 pix.
= 302.624 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9457 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.805
Minimum - Maximum-5.8679824 - 7.9438653
Average (Standard dev.)-0.0007376312 (±0.18718572)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 302.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_50058_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_50058_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Octameric Fav pore in complex with sphingomyelin and cholesterol ...

EntireName: Octameric Fav pore in complex with sphingomyelin and cholesterol molecules solubilized by detergents
Components
  • Complex: Octameric Fav pore in complex with sphingomyelin and cholesterol molecules solubilized by detergents
    • Protein or peptide: Actinoporin
  • Ligand: CHOLESTEROL
  • Ligand: Sphingomyelin C18

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Supramolecule #1: Octameric Fav pore in complex with sphingomyelin and cholesterol ...

SupramoleculeName: Octameric Fav pore in complex with sphingomyelin and cholesterol molecules solubilized by detergents
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Octameric Fav pore prepared on 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC):sphingomyelin:cholesterol (1:1:1 molar ratio) membranes
Source (natural)Organism: Orbicella faveolata (invertebrata)

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Macromolecule #1: Actinoporin

MacromoleculeName: Actinoporin / type: protein_or_peptide / ID: 1
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).
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Orbicella faveolata (invertebrata)
Molecular weightTheoretical: 28.80326 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GHMSAPIKAN DPNGEVLEEM PKTKRGAEAL LADVGLAHFP EMVPNRQELR ALLKRSHNAE AIPQEPMDLE NLDSEKRAAR IAAGTIIAG AELTIGLLQN LLDVLANVNR KCAVGVDNES GFRWQEGSTY FFSGTADENL PYSVSDGYAV LYGPRKTNGP V ATGVVGVL ...String:
GHMSAPIKAN DPNGEVLEEM PKTKRGAEAL LADVGLAHFP EMVPNRQELR ALLKRSHNAE AIPQEPMDLE NLDSEKRAAR IAAGTIIAG AELTIGLLQN LLDVLANVNR KCAVGVDNES GFRWQEGSTY FFSGTADENL PYSVSDGYAV LYGPRKTNGP V ATGVVGVL AYYIPSIGKT LAVMWSVPFD YNFYQNWWNA KLYSGNQDAD YDHYVDLYYD ANPFKANGWH ERSLGSGLKF CG SMSSSGQ ATLEIHVLKE SETCM

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 32 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #3: Sphingomyelin C18

MacromoleculeName: Sphingomyelin C18 / type: ligand / ID: 3 / Number of copies: 72 / Formula: A1H8M
Molecular weightTheoretical: 732.089 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
50.0 mMTris
0.02 %Brij 35

Details: 150 mM NaCl, 50 mM Tris/HCl, 0.02 % Brij 35, pH 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1266343 / Details: Number of particles after template picking
CTF correctionSoftware - Name: cryoSPARC (ver. v4) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C8 (8 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4) / Number images used: 223527
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. V4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. v4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model
Details: Pore structure of the same protein from related entries
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9eyn:
The structure of solubilized octameric pore of actinoporin Fav prepared on DOPC, cholesterol, sphingomyelin membranes

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