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- PDB-6bhu: Cryo-EM structure of ATP-bound, outward-facing bovine multidrug r... -

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Basic information

Entry
Database: PDB / ID: 6bhu
TitleCryo-EM structure of ATP-bound, outward-facing bovine multidrug resistance protein 1 (MRP1)
ComponentsMultidrug resistance-associated protein 1
KeywordsTRANSPORT PROTEIN / ABC transporter / multidrug resistance / outward facing
Function / homologyMulti drug resistance-associated protein / ABC transporter integral membrane type-1 fused domain profile. / ABC transporters family signature. / ABC transporter transmembrane region / ABC transporter / ABC transporter type 1, transmembrane domain superfamily / P-loop containing nucleoside triphosphate hydrolase / ABC transporter, conserved site / ABC transporter type 1, transmembrane domain / AAA+ ATPase domain ...Multi drug resistance-associated protein / ABC transporter integral membrane type-1 fused domain profile. / ABC transporters family signature. / ABC transporter transmembrane region / ABC transporter / ABC transporter type 1, transmembrane domain superfamily / P-loop containing nucleoside triphosphate hydrolase / ABC transporter, conserved site / ABC transporter type 1, transmembrane domain / AAA+ ATPase domain / ABC transporter-like / ATP-binding cassette, ABC transporter-type domain profile. / glutathione transmembrane transport / glutathione transmembrane transporter activity / glutathione S-conjugate-exporting ATPase activity / xenobiotic transport / xenobiotic transmembrane transporting ATPase activity / drug transmembrane transport / basolateral plasma membrane / integral component of plasma membrane / ATP binding / Multidrug resistance-associated protein 1
Function and homology information
Specimen sourceBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.14 Å resolution
AuthorsJohnson, Z.L. / Chen, J.
CitationJournal: Cell / Year: 2018
Title: ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1.
Authors: Zachary Lee Johnson / Jue Chen
Abstract: The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site ...The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 31, 2017 / Release: Dec 27, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 27, 2017Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure modelDatabase referencescitation / pdbx_database_related_citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _pdbx_database_related.content_type
1.2Jan 24, 2018Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
1.3Oct 3, 2018Structure modelData collection / Refinement descriptionrefine

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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)185,2978
Polyers183,0741
Non-polymers2,2237
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Multidrug resistance-associated protein 1 / ATP-binding cassette sub-family C member 1 / Leukotriene C(4) transporter / LTC4 transporter


Mass: 183074.062 Da / Num. of mol.: 1 / Mutation: E1454Q / Source: (gene. exp.) Bos taurus (cattle) / Gene: ABCC1, MRP1 / Cell line (production host): HEK293S GntI- / Production host: Homo sapiens (human) / References: UniProt: Q8HXQ5
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Formula: Mg / Magnesium
#4: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Formula: C27H46O / Cholesterol
Sequence detailsRESIDUES 30-194, CORRESPONDING TO TMD0, ARE MODELED AS POLY- ALANINE (POLY UNK). THE REGISTER IN ...RESIDUES 30-194, CORRESPONDING TO TMD0, ARE MODELED AS POLY- ALANINE (POLY UNK). THE REGISTER IN THIS REGION COULD NOT BE CONFIDENTLY ESTABLISHED AND THUS THE NUMBERING ASSIGNED TO THE RESIDUES IS PUTATIVE. THE POLY-ALANINE REGIONS HAVE BEEN RENAMED AS UNKNOW

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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

ComponentName: bovine multidrug resistance protein 1 (MRP1) E1454Q / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Cell: HEK293S GntI- / Organism: Homo sapiens (human) / 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
780 uMLeukotriene C41
82.5 %DMSO1
SpecimenConc.: 4.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
Grid type: Quantifoil R1.2/1.3 400-mesh Au Holey Carbon Grids
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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 / Nominal magnification: 37000 / Nominal defocus max: 2400 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 kelvins / Temperature (min): 80 kelvins
Image recordingAverage exposure time: 7 sec. / Electron dose: 84 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 4210
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7Coot0.8.7model fitting
9RELION1.4initial Euler assignment
10FREALIGNfinal Euler assignment
12FREALIGN3D reconstruction
13PHENIX1.11model refinement
14REFMACCCP4 7.0model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 354752
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 354752 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: RECIPROCAL
RefineCorrelation coeff Fo to Fc: 0.969 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS / Overall ESU R: 0.43
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Solvent computationSolvent ion probe radii: 0.8 Å / Solvent shrinkage radii: 0.8 Å / Solvent vdw probe radii: 1.2 Å / Solvent model details: MASK
Displacement parametersB iso mean: 204.276 Å2 / Aniso B11: -3.4 Å2 / Aniso B12: -1.23 Å2 / Aniso B13: 1.08 Å2 / Aniso B22: 2.55 Å2 / Aniso B23: -0.8 Å2 / Aniso B33: 0.84 Å2
Least-squares processR factor R work: 0.29701 / R factor obs: 0.29701 / Highest resolution: 3.14 Å / Lowest resolution: 142 Å / Number reflection obs: 89483 / Percent reflection obs: 99.99
Number of atoms included #1Total: 10273
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01910475
ELECTRON MICROSCOPYr_bond_other_d0.0000.02010096
ELECTRON MICROSCOPYr_angle_refined_deg1.1051.97614258
ELECTRON MICROSCOPYr_angle_other_deg3.7593.00023284
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.0535.0001319
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.28023.856402
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.38415.0001721
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.79615.00056
ELECTRON MICROSCOPYr_chiral_restr0.0580.2001694
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.02011472
ELECTRON MICROSCOPYr_gen_planes_other0.0080.0202113
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_it4.02721.5055303
ELECTRON MICROSCOPYr_mcbond_other4.02721.5045302
ELECTRON MICROSCOPYr_mcangle_it6.94532.2256613
ELECTRON MICROSCOPYr_mcangle_other6.94532.2266614
ELECTRON MICROSCOPYr_scbond_it4.56520.3185172
ELECTRON MICROSCOPYr_scbond_other4.56520.3175173
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other7.92430.5797646
ELECTRON MICROSCOPYr_long_range_B_refined13.73512476
ELECTRON MICROSCOPYr_long_range_B_other13.73412477
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 3.14 Å / R factor R work: 0.819 / Lowest resolution: 3.222 Å / Number reflection R free: 0 / Number reflection R work: 6640 / Total number of bins used: 20 / Percent reflection obs: 1

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