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- PDB-6uy0: Cryo-EM structure of wild-type bovine multidrug resistance protei... -

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Basic information

Entry
Database: PDB / ID: 6uy0
TitleCryo-EM structure of wild-type bovine multidrug resistance protein 1 (MRP1) under active turnover conditions
ComponentsMultidrug resistance-associated protein 1
KeywordsTRANSPORT PROTEIN / ABC transporter / multidrug resistance / outward facing / MRP1
Function / homology
Function and homology information


Sphingolipid de novo biosynthesis / Heme degradation / Synthesis of Leukotrienes (LT) and Eoxins (EX) / cyclic nucleotide transport / Transport of RCbl within the body / Paracetamol ADME / leukotriene transport / glutathione transmembrane transport / glutathione transmembrane transporter activity / ABC-family proteins mediated transport ...Sphingolipid de novo biosynthesis / Heme degradation / Synthesis of Leukotrienes (LT) and Eoxins (EX) / cyclic nucleotide transport / Transport of RCbl within the body / Paracetamol ADME / leukotriene transport / glutathione transmembrane transport / glutathione transmembrane transporter activity / ABC-family proteins mediated transport / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / Cytoprotection by HMOX1 / ABC-type xenobiotic transporter / ABC-type xenobiotic transporter activity / lipid transport / xenobiotic transport / xenobiotic transmembrane transporter activity / ABC-type transporter activity / positive regulation of inflammatory response / basolateral plasma membrane / response to xenobiotic stimulus / ATP hydrolysis activity / ATP binding
Similarity search - Function
Multi drug resistance-associated protein / : / 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 ...Multi drug resistance-associated protein / : / 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'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / CHOLESTEROL / Multidrug resistance-associated protein 1
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsJohnson, Z.L. / Chen, J.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2020
Title: Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses.
Authors: Ling Wang / Zachary Lee Johnson / Michael R Wasserman / Jesper Levring / Jue Chen / Shixin Liu /
Abstract: ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles ...ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.
History
DepositionNov 9, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Multidrug resistance-associated protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)175,0068
Polymers172,8631
Non-polymers2,1437
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Multidrug resistance-associated protein 1 / ATP-binding cassette sub-family C member 1 / Glutathione-S-conjugate-translocating ATPase ABCC1 / ...ATP-binding cassette sub-family C member 1 / Glutathione-S-conjugate-translocating ATPase ABCC1 / Leukotriene C(4) transporter / LTC4 transporter


Mass: 172862.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: ABCC1, MRP1 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human)
References: UniProt: Q8HXQ5, ABC-type xenobiotic transporter, ABC-type glutathione-S-conjugate transporter
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
Has ligand of interestY

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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: bovine multidrug resistance protein 1 (MRP1) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GntI- / Plasmid: Baculovirus
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
1150 mMKCl1
250 mMTris1
32 mMMgCl21
42 mMDTT1
50.06 %Digitonin1
63 mMFos-Choline-8, fluorinated1
710 mMATP-Mg1
880 uMLeukotriene C41
92.5 %DMSO1
SpecimenConc.: 5.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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 FIELD / Nominal magnification: 29000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 75 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3993
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: REFMAC / Version: 5.8.0253 / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Coot0.8.7model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.11model refinement
14REFMACCCP4 7.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1143729
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81078 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 6BHU

6bhu


Accession code: 6BHU / Source name: PDB / Type: experimental model
RefinementResolution: 3.23→143 Å / Cor.coef. Fo:Fc: 0.895 / ESU R: 0.61
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34282 --
obs0.34282 70693 99.99 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 139.941 Å2
Baniso -1Baniso -2Baniso -3
1--2.89 Å2-1.75 Å20.01 Å2
2--1.21 Å2-0.35 Å2
3---1.68 Å2
Refinement stepCycle: 1 / Total: 9684
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.0139892
ELECTRON MICROSCOPYr_bond_other_d0.0350.0179506
ELECTRON MICROSCOPYr_angle_refined_deg1.2971.62713450
ELECTRON MICROSCOPYr_angle_other_deg2.2921.56721990
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.22351206
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.50522.078462
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.363151719
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.1461556
ELECTRON MICROSCOPYr_chiral_restr0.0520.21315
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0210753
ELECTRON MICROSCOPYr_gen_planes_other0.0030.022049
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.60414.9424836
ELECTRON MICROSCOPYr_mcbond_other3.60414.9414835
ELECTRON MICROSCOPYr_mcangle_it5.94922.4346038
ELECTRON MICROSCOPYr_mcangle_other5.94822.4356039
ELECTRON MICROSCOPYr_scbond_it3.45715.3685056
ELECTRON MICROSCOPYr_scbond_other3.45715.3695057
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other5.9622.9297413
ELECTRON MICROSCOPYr_long_range_B_refined9.87711476
ELECTRON MICROSCOPYr_long_range_B_other9.87711477
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.23→3.314 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.951 5204 -
obs--99.94 %

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