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

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Basic information

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
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsAlam, A.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
European Molecular Biology Organization Switzerland
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
History
DepositionJan 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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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
Polymers192,3303
Non-polymers5,17313
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11680 Å2
ΔGint-35 kcal/mol
Surface area74240 Å2
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 method: isolated from a genetically manipulated source
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 method: isolated from a genetically manipulated source
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 method: isolated from a genetically manipulated source
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 / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
#5: Chemical ChemComp-ZQU / Zosuquidar / Zosuquidar


Mass: 527.604 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H31F2N3O2
#6: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


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


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

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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

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Nanodisc reconstituted ABCB1HM (human mouse chimeric ABCB1) EQ mutant in complex with UIC2 Fab and zosuquidar1, 2, 30MULTIPLE SOURCES
2ABCB1HM11RECOMBINANT
3UIC2 Fab2,31RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
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 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291197 / Symmetry type: POINT
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.010313274
f_angle_d1.187918029
f_chiral_restr0.06412069
f_plane_restr0.00772227
f_dihedral_angle_d13.56557775

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