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- PDB-8sa0: CryoEM structure of P-Glycoprotein in occluded closed state under... -

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Basic information

Entry
Database: PDB / ID: 8sa0
TitleCryoEM structure of P-Glycoprotein in occluded closed state under continuous turnover conditions with verapamil
ComponentsATP-dependent translocase ABCB1
KeywordsTRANSPORT PROTEIN / Multidrug Resistance / ABC Transporter / Membrane Protein / Transporter.
Function / homology
Function and homology information


terpenoid transport / ceramide floppase activity / phosphatidylethanolamine floppase activity / carboxylic acid transmembrane transport / regulation of chloride transport / floppase activity / ceramide translocation / Abacavir transmembrane transport / carboxylic acid transmembrane transporter activity / phosphatidylethanolamine flippase activity ...terpenoid transport / ceramide floppase activity / phosphatidylethanolamine floppase activity / carboxylic acid transmembrane transport / regulation of chloride transport / floppase activity / ceramide translocation / Abacavir transmembrane transport / carboxylic acid transmembrane transporter activity / phosphatidylethanolamine flippase activity / phosphatidylcholine floppase activity / xenobiotic transport across blood-brain barrier / stem cell proliferation / external side of apical plasma membrane / Atorvastatin ADME / export across plasma membrane / P-type phospholipid transporter / transepithelial transport / xenobiotic detoxification by transmembrane export across the plasma membrane / ABC-type xenobiotic transporter / phospholipid translocation / Prednisone ADME / ABC-type xenobiotic transporter activity / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / transmembrane transporter activity / xenobiotic metabolic process / G2/M transition of mitotic cell cycle / ABC-family protein mediated transport / transmembrane transport / 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 / Dexverapamil / 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
DepositionMar 30, 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 1.1Jun 4, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 4, 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)142,5196
Polymers141,0011
Non-polymers1,5185
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, negative stain EM
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: 141001.062 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-I6H / Dexverapamil


Mass: 454.602 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H38N2O4 / 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: P-Glycoprotein in occluded closed state under continuous turnover conditions with verapamil
Type: CELL / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
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 defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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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: 49787 / 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_d0.0035860
ELECTRON MICROSCOPYf_angle_d0.4938173
ELECTRON MICROSCOPYf_dihedral_angle_d7.8831249
ELECTRON MICROSCOPYf_chiral_restr0.0451068
ELECTRON MICROSCOPYf_plane_restr0.0061181

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