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- PDB-8sb9: CryoEM structure of P-Glycoprotein in inward facing 1 state under... -

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Basic information

Entry
Database: PDB / ID: 8sb9
TitleCryoEM structure of P-Glycoprotein in inward facing 1 state under continuous turnover conditions with vinblastine
ComponentsATP-dependent translocase ABCB1
KeywordsTRANSPORT PROTEIN / multidrug resistance / ABC transporter / membrane protein / transporter
Function / homology
Function and homology information


carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / hormone transport / cellular response to nonylphenol / cellular response to borneol / response to codeine / response to cyclosporin A / cellular response to mycotoxin / daunorubicin transport / positive regulation of response to drug ...carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / hormone transport / cellular response to nonylphenol / cellular response to borneol / response to codeine / response to cyclosporin A / cellular response to mycotoxin / daunorubicin transport / positive regulation of response to drug / terpenoid transport / ceramide floppase activity / negative regulation of sensory perception of pain / positive regulation of establishment of Sertoli cell barrier / regulation of intestinal absorption / cellular response to external biotic stimulus / response to quercetin / response to antineoplastic agent / ceramide translocation / floppase activity / Abacavir transmembrane transport / establishment of blood-retinal barrier / phosphatidylethanolamine flippase activity / protein localization to bicellular tight junction / external side of apical plasma membrane / phosphatidylcholine floppase activity / Atorvastatin ADME / xenobiotic transport across blood-brain barrier / response to thyroxine / establishment of blood-brain barrier / transepithelial transport / xenobiotic detoxification by transmembrane export across the plasma membrane / export across plasma membrane / P-type phospholipid transporter / ABC-type xenobiotic transporter / cellular response to L-glutamate / response to vitamin A / response to vitamin D / response to glycoside / ABC-type xenobiotic transporter activity / response to glucagon / intestinal absorption / response to alcohol / Prednisone ADME / phospholipid translocation / cellular response to antibiotic / cellular hyperosmotic salinity response / maintenance of blood-brain barrier / cellular response to alkaloid / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / cellular response to dexamethasone stimulus / response to cadmium ion / transport across blood-brain barrier / lactation / xenobiotic metabolic process / regulation of chloride transport / response to progesterone / placenta development / stem cell proliferation / ABC-family proteins mediated transport / cellular response to estradiol stimulus / brush border membrane / female pregnancy / circadian rhythm / transmembrane transport / G2/M transition of mitotic cell cycle / cellular response to tumor necrosis factor / cellular response to lipopolysaccharide / response to hypoxia / apical plasma membrane / response to xenobiotic stimulus / ubiquitin protein ligase binding / cell surface / ATP hydrolysis activity / extracellular exosome / ATP binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Chem-VLB / ATP-dependent translocase ABCB1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCulbertson, A. / Liao, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM of human P-glycoprotein reveals an intermediate occluded conformation during active drug transport.
Authors: Alan T Culbertson / Maofu Liao /
Abstract: P-glycoprotein (Pgp) is an important human multidrug transporter that contributes to pharmacokinetics and multidrug resistance. Despite decades of study, the conformation transition cycle of Pgp ...P-glycoprotein (Pgp) is an important human multidrug transporter that contributes to pharmacokinetics and multidrug resistance. Despite decades of study, the conformation transition cycle of Pgp undergoing active drug transport is not defined, thus the precise relevance of all available Pgp structures to uninterrupted multidrug transport remains unclear. Here, we use cryo-EM of membrane-embedded human Pgp under continuous turnover conditions to analyze the conformational ensembles of Pgp transporting distinct substrates. These results delineate multiple conformations including inward-facing and closed conformations, highlighting the occluded conformation as a critical intermediate state between transporter closure and substrate release. A combination of structural, functional, and computational studies reveals the transmembrane helices 4 and 10 undergoing drastic rearrangement to coordinate substrate binding, occlusion, and release, and identifies a peripheral site involved in substrate capture and Pgp inhibition. Together, our results provide a set of snapshots of Pgp undergoing continuous drug transport, unveiling the intricate interplay between transporter dynamics and drug movement, and shed light on the mechanism of polyspecificity.
History
DepositionApr 2, 2023Deposition site: RCSB / Processing site: RCSB
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: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATP-dependent translocase ABCB1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)144,3307
Polymers141,6451
Non-polymers2,6856
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ATP-dependent translocase ABCB1 / ATP-binding cassette sub-family B member 1 / Multidrug resistance protein 1 / P-glycoprotein 1 / ...ATP-binding cassette sub-family B member 1 / Multidrug resistance protein 1 / P-glycoprotein 1 / Phospholipid transporter ABCB1


Mass: 141644.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCB1, MDR1, PGY1 / Production host: Homo sapiens (human)
References: UniProt: P08183, ABC-type xenobiotic transporter, P-type phospholipid transporter
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-VLB / (2ALPHA,2'BETA,3BETA,4ALPHA,5BETA)-VINCALEUKOBLASTINE / VINBLASTINE


Mass: 810.974 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C46H58N4O9 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: CryoEM structure of P-Glycoprotein in inward facing 1 state under continuous turnover conditions with vinblastine
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2150 mMSodium ChlorideNaCl1
30.1 mMTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9372

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32832 / Symmetry type: POINT
RefinementHighest resolution: 4.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037524
ELECTRON MICROSCOPYf_angle_d0.52510404
ELECTRON MICROSCOPYf_dihedral_angle_d11.2522068
ELECTRON MICROSCOPYf_chiral_restr0.041282
ELECTRON MICROSCOPYf_plane_restr0.0041348

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