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- EMDB-49369: Tuna P-glycoprotein Apo Conformation 4 -

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Basic information

Entry
Database: EMDB / ID: EMD-49369
TitleTuna P-glycoprotein Apo Conformation 4
Map data
Sample
  • Complex: ABC transporter from yellowfin tuna
    • Protein or peptide: Permeability Glycoprotein (P-gp)
KeywordsABC Transporter / Membrane protein / TRANSPORT PROTEIN
Biological speciesThunnus albacares (yellowfin tuna)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsYoung MA / Rees SD / Nicklisch SCT / Stowell M / Hamdoun A / Chang G
Funding support United States, 1 items
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, 2025-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49369.png

Downloads & links

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Map

FileDownload / File: emd_49369.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.87 Å/pix.
x 160 pix.
= 299.2 Å		1.87 Å/pix.
x 160 pix.
= 299.2 Å		1.87 Å/pix.
x 160 pix.
= 299.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-1.3343064 - 1.852495
Average (Standard dev.)0.0004873764 (±0.04679739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 299.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49369_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_49369_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ABC transporter from yellowfin tuna

EntireName: ABC transporter from yellowfin tuna
Components
  • Complex: ABC transporter from yellowfin tuna
    • Protein or peptide: Permeability Glycoprotein (P-gp)

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Supramolecule #1: ABC transporter from yellowfin tuna

SupramoleculeName: ABC transporter from yellowfin tuna / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thunnus albacares (yellowfin tuna)
Molecular weightTheoretical: 1.43 MDa

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Macromolecule #1: Permeability Glycoprotein (P-gp)

MacromoleculeName: Permeability Glycoprotein (P-gp) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thunnus albacares (yellowfin tuna)
Molecular weightTheoretical: 144.6805 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MEGKEEMEML ANAKPHKNGG LREVKDEDDK KTGEKKKEKG AKLPMVGPLA LFRFADGKDI VLIFMGTVMS VAHGAVLPLM CIVFGDMTD SFIKDSMTSH IQITQITNPQ FTFTYPQSTL QEDMQSFAIY YSIMGFVVLV AAYMQVSFWA LAAGRQVRRI R KLFFHRIM ...String:
MEGKEEMEML ANAKPHKNGG LREVKDEDDK KTGEKKKEKG AKLPMVGPLA LFRFADGKDI VLIFMGTVMS VAHGAVLPLM CIVFGDMTD SFIKDSMTSH IQITQITNPQ FTFTYPQSTL QEDMQSFAIY YSIMGFVVLV AAYMQVSFWA LAAGRQVRRI R KLFFHRIM QQDIGWFDVN ETGELNTRLT DDVYKIQEGI GDKVGMLIQA FSTFITSFII GFVKGWKLTL VILAVSPALG IS AAIFGKV LTNFTAKEQT AYAKAGAVAE EVLSAIRTVF AFSGQDREIK RYHKNLEDAK NMGIKKAISA NIAMGFTFLM IYL SYALAF WYGSILIMSK EYTIGTVLTV FFVVLIGAFT MGQTSPNIQS FASARGAAHK VYSIIDNQPC IDSYSDAGFK PDSI KGNIE FRNIHFNYPS RPDVKILNNM SLSVKSGQTI ALVGSSGCGK STTIQLLQRF YDPQEGSVSI DTHDIRSLNV RYLRE MIGV VSQEPILFAT TIAENIKYGR PDVTQQEIEQ AAKEANAYDF IMNLPDKFET LVGDRGTQMS GGQKQRIAIA RALVRN PKI LLLDEATSAL DAESETIVQA ALDKVRLGRT TIVVAHRLST IRNADVIAGF HKGDVIELGT HSQLMEKQGV YYTLVTM QT FQQVEDGEES EYEQAEDEKS PSVKSFSQSS LYRRKSTRGS SFAGSEGERE EKEKLRDVTD RAEEDENVPP VSFLKVMR L NLSEWPYMAL GTFCAIINGM MQPLFAVIFS KIIAVFAEPN QEIVRQKSEF FSLMFAAIGG VTFVTMFLQG FCFGKSGEL LTLKLRLGAF KSMMRQDLGW FDNPKNSVGA LTTRLATDAA QVQGATGVRM ATLAQNIANL GTSIIISFVY GWELTLLILS VVPIMAVAG SVQMQLLAGH AAEDKKELEK AGKIATEAIE NIRTVASLTR EPKFESLYQE NLHVPYKNSQ KKAHVYGFTF S FSQAMIYF AYAGCFRFGA WLIKEGRMDA EGVYLVISAV LFGAMAVGEA NSFTPNYAKA KMSASHLMML MNREPAIDNL SE EGQSPDK FDGNVRFEGV KFNYPSRPEV PILRGLNLKV SKGETLALVG SSGCGKSTTI QLLERFYDPM HGKVELDGIS AKQ LNIHWL RSQIGIVSQE PVLFDCTLAE NIAYGDNSRT VTLEEIQAAA KAANIHSFIE NLPQGYDTQA GDKGTQLSGG QKQR IAIAR AILRNPKLLL LDEATSALDT ESEKVVQEAL DQASRGRTCI VVAHRLSTIQ NADRIAVFQA GVVVEQGTHQ QLLAK KGIY SMLVNTQMGH ERNCIDHHHH HH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHEPES
150.0 mMSodium chlorideNaCl
1.0 mMMagnesium chlorideMgCl2
0.02 %LMNG

Details: 50 mM HEPES pH 8, 150 mM NaCl, 0.02% LMNG, 1mM MgCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: SPT LABTECH CHAMELEON
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..
DetailsVitrified, monodisperse sample of transporter proteins in detergent micelles

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4800000
CTF correctionSoftware - Name: cryoSPARC (ver. v4.5.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: AlphaFold 2 Model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 82929
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The initial model was predicted using AlphaFold 2 software.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 127
Output model

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

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