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- PDB-6qee: Nanodisc reconstituted Human-mouse chimeric ABCB1 (ABCB1HM)-EQ mu... -

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Entry
Database: PDB / ID: 6qee
TitleNanodisc reconstituted Human-mouse chimeric ABCB1 (ABCB1HM)-EQ mutant in complex with UIC2 Fab and Zosuquidar.
Components
  • (UIC2 Antigen Binding Fragment ...) x 2
  • ABCB1HM-EQ
KeywordsMEMBRANE PROTEIN / ABCB1 / p-glycoprotein / p-gp / multidrug transporter / ABC transporter / zosuquidar / membrane transporter
Specimen sourceHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsAlam, A.
CitationJournal: Science / Year: 2019
Title: Structural insight into substrate and inhibitor discrimination by human P-glycoprotein.
Authors: Amer Alam / Julia Kowal / Eugenia Broude / Igor Roninson / Kaspar P Locher
Abstract: ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic ...ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo-electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate-hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 7, 2019 / Release: Feb 27, 2019

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Structure visualization

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Assembly

Deposited unit
A: ABCB1HM-EQ
B: UIC2 Antigen Binding Fragment Light chain
C: UIC2 Antigen Binding Fragment Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,50416
Polyers192,3303
Non-polymers5,17313
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)11680
ΔGint (kcal/M)-35
Surface area (Å2)74240
MethodPISA

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Components

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Protein/peptide , 1 types, 1 molecules A

#1: Protein/peptide ABCB1HM-EQ


Mass: 143627.828 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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UIC2 Antigen Binding Fragment ... , 2 types, 2 molecules BC

#2: Protein/peptide UIC2 Antigen Binding Fragment Light chain


Mass: 24321.039 Da / Num. of mol.: 1 / Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Protein/peptide UIC2 Antigen Binding Fragment Heavy Chain


Mass: 24381.281 Da / Num. of mol.: 1 / Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Non-polymers , 4 types, 13 molecules

#4: Chemical ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 3 / Formula: C8H15NO6 / N-Acetylglucosamine
#5: Chemical ChemComp-ZQU / Zosuquidar


Mass: 527.604 Da / Num. of mol.: 2 / Formula: C32H31F2N3O2 / Zosuquidar
#6: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 7 / Formula: C27H46O / Cholesterol
#7: Chemical ChemComp-3PE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE / 3-SN-PHOSPHATIDYLETHANOLAMINE


Mass: 748.065 Da / Num. of mol.: 1 / Formula: C41H82NO8P / Comment: phospholipid *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Nanodisc reconstituted ABCB1HM (human mouse chimeric ABCB1) EQ mutant in complex with UIC2 Fab and zosuquidarCOMPLEX1, 2, 30MULTIPLE SOURCES
2ABCB1HMCOMPLEX11RECOMBINANT
3UIC2 FabCOMPLEX2,31RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129606Homo sapiens (human)
2310090Mus musculus (house mouse)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
129606Homo sapiens (human)
2310090Mus musculus (house mouse)
Buffer solutionpH: 7.5
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 2.1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 291197 / Symmetry type: POINT
RefineStereochemistry target values: CDL v1.2
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010313274
ELECTRON MICROSCOPYf_angle_d1.187918029
ELECTRON MICROSCOPYf_chiral_restr0.06412069
ELECTRON MICROSCOPYf_plane_restr0.00772227
ELECTRON MICROSCOPYf_dihedral_angle_d13.56557775

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