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- PDB-9eyl: dN53 deletion variant of monomeric Fav - actinoporin from Orbicel... -

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Basic information

Entry
Database: PDB / ID: 9eyl
TitledN53 deletion variant of monomeric Fav - actinoporin from Orbicella faveolata
ComponentsdN53_Fav
KeywordsTOXIN / Actinoporin / Pore-forming toxin / Orbicella faveolata
Function / homologyTRIS-HYDROXYMETHYL-METHYL-AMMONIUM
Function and homology information
Biological speciesOrbicella faveolata (invertebrata)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsSolinc, G. / Svigelj, T. / Anderluh, G. / Podobnik, M.
Funding support Slovenia, United Kingdom, 3items
OrganizationGrant numberCountry
Slovenian Research AgencyJ4-8225 Slovenia
Slovenian Research AgencyP1-0391 Slovenia
Other private United Kingdom
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 1.1Nov 5, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: dN53_Fav
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3174
Polymers23,0031
Non-polymers3143
Water4,936274
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area520 Å2
ΔGint-16 kcal/mol
Surface area8170 Å2
Unit cell
Length a, b, c (Å)61.011, 61.011, 91.825
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Components on special symmetry positions
IDModelComponents
11A-567-

HOH

21A-621-

HOH

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Components

#1: Protein dN53_Fav


Mass: 23002.561 Da / Num. of mol.: 1 / Mutation: dN53
Source method: isolated from a genetically manipulated source
Details: This protein was expressed with an N-terminal deletion of 53 residues compared to the wild type. The deletion construct has three additional residues at the N-terminal (GHM) from the expression system.
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-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: SO4
#3: Chemical ChemComp-144 / TRIS-HYDROXYMETHYL-METHYL-AMMONIUM


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 274 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.14 Å3/Da / Density % sol: 42.65 % / Description: hexagonal rods
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop / Details: 1.8 M Li2SO4 / PH range: 6-8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ELETTRA / Beamline: 5.2R / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: May 31, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.5→45.8 Å / Num. obs: 32274 / % possible obs: 99.95 % / Redundancy: 10.4 % / Biso Wilson estimate: 11.89 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.09948 / Rpim(I) all: 0.03232 / Rrim(I) all: 0.1047 / Net I/σ(I): 18.73
Reflection shellResolution: 1.5→1.554 Å / Redundancy: 10.1 % / Rmerge(I) obs: 0.7893 / Mean I/σ(I) obs: 3.25 / Num. unique obs: 3195 / CC1/2: 0.887 / CC star: 0.969 / Rpim(I) all: 0.26 / Rrim(I) all: 0.8317 / % possible all: 99.59

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
XDS0.76data reduction
XDS0.76data scaling
PHASER(1.20.1_4487: ???)phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.5→45.8 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 17.9 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1846 1978 6.13 %
Rwork0.1602 --
obs0.1617 32269 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.5→45.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1424 0 18 274 1716
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0071550
X-RAY DIFFRACTIONf_angle_d0.8862124
X-RAY DIFFRACTIONf_dihedral_angle_d6.905223
X-RAY DIFFRACTIONf_chiral_restr0.059223
X-RAY DIFFRACTIONf_plane_restr0.005276
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.540.32811380.23132141X-RAY DIFFRACTION99
1.54-1.580.22171350.19582125X-RAY DIFFRACTION100
1.58-1.630.22561400.18262133X-RAY DIFFRACTION100
1.63-1.680.22811390.16982135X-RAY DIFFRACTION100
1.68-1.740.19031430.17632142X-RAY DIFFRACTION100
1.74-1.810.21141380.16782118X-RAY DIFFRACTION100
1.81-1.890.20031360.17512162X-RAY DIFFRACTION100
1.89-1.990.19461400.15572139X-RAY DIFFRACTION100
1.99-2.110.17861390.1522165X-RAY DIFFRACTION100
2.11-2.280.15241410.15562147X-RAY DIFFRACTION100
2.28-2.510.19311430.16722176X-RAY DIFFRACTION100
2.51-2.870.17871440.16022186X-RAY DIFFRACTION100
2.87-3.610.15911450.14822201X-RAY DIFFRACTION100
3.62-45.80.16941570.14342321X-RAY DIFFRACTION100

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