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- PDB-9nfl: Tuna P-glycoprotein Apo Conformation 4 -

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Basic information

Entry
Database: PDB / ID: 9nfl
TitleTuna P-glycoprotein Apo Conformation 4
ComponentsPermeability Glycoprotein (P-gp)
KeywordsTRANSPORT PROTEIN / ABC Transporter / Membrane protein
Biological speciesThunnus albacares (yellowfin tuna)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsYoung, M.A. / Rees, S.D. / Nicklisch, S.C.T. / Stowell, M. / Hamdoun, A. / Chang, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ES027921 United States
CitationJournal: Environ Sci Technol / Year: 2025
Title: Cryo-EM Structures of Apo and DDT-Bound P-Glycoprotein in Yellowfin Tuna.
Authors: Megan A Young / Steven D Rees / Sascha C T Nicklisch / Michael H B Stowell / Amro Hamdoun / Geoffrey Chang /
Abstract: Persistent pollutants in the ocean impact the safety of seafood. Many emerging and legacy persistent organic pollutants (POPs) have been disposed into the world's oceans, exemplified by the recent ...Persistent pollutants in the ocean impact the safety of seafood. Many emerging and legacy persistent organic pollutants (POPs) have been disposed into the world's oceans, exemplified by the recent discovery of large amounts of the halogenated pesticide dichlorodiphenyltrichloroethane (DDT) waste in the waters of Southern California. The biological mechanisms governing persistence and trophic transfer of marine pollutants into seafood species remain incompletely understood. Xenobiotic transporters, such as P-glycoprotein (P-gp), are present in all organisms and prevent the accumulation of toxic chemicals. Our previous work has demonstrated that halogenated marine pollutants can act as inhibitors of human and murine P-gp transporters by interacting with their binding site and impeding transport. Using cryo-EM, we determined the molecular interactions of DDT with P-glycoprotein from yellowfin tuna (). The results reveal that the conformation of the transporter samples multiple degrees of widening in the absence of substrate. We also show that DDT binds in a singular, wide inward-facing conformation that could inhibit the transport cycle. This transporter inhibition may contribute to the bioaccumulation of DDT in tuna. This study highlights the capacity of persistent organic pollutants to act at multiple points in the food chain to inhibit this critical transport mechanism.
History
DepositionFeb 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Permeability Glycoprotein (P-gp)


Theoretical massNumber of molelcules
Total (without water)144,6811
Polymers144,6811
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Permeability Glycoprotein (P-gp)


Mass: 144680.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thunnus albacares (yellowfin tuna) / Production host: Komagataella pastoris (fungus)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ABC transporter from yellowfin tuna / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.43 MDa / Experimental value: NO
Source (natural)Organism: Thunnus albacares (yellowfin tuna)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 8
Details: 50 mM HEPES pH 8, 150 mM NaCl, 0.02% LMNG, 1mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2150 mMSodium chlorideNaCl1
31 mMMagnesium chlorideMgCl21
40.02 %LMNG1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Vitrified, monodisperse sample of transporter proteins in detergent micelles
VitrificationInstrument: SPT LABTECH CHAMELEON / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: The grids were glow discharged for 80sec at 12mA, with a relative humidity of 82% and temperature 21.5oC maintained throughout preparation. Samples of purified protein were concentrated to ...Details: The grids were glow discharged for 80sec at 12mA, with a relative humidity of 82% and temperature 21.5oC maintained throughout preparation. Samples of purified protein were concentrated to 1mg/mL and ultracentrifuged in a Beckmann Optima Ultracentrifuge for 15 minutes prior to grid preparation. The sample was applied via 2-S application mode by the Chameleon, allowed to wick for 530ms via the grid nanowires, and subsequently plunged into liquid ethane maintained at -180oC.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.5.3particle selection
2cryoSPARCv4.5.3image acquisition
4cryoSPARCv4.5.3CTF correction
7PHENIX1.21.1_5286model fitting
9PHENIX1.21.1_5286model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4800000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82929 / Symmetry type: POINT
Atomic model buildingB value: 127 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: The initial model was predicted using AlphaFold 2 software.
Source name: AlphaFold / Type: in silico model

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