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- EMDB-20945: Cryo-EM structure of wild-type bovine multidrug resistance protei... -

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Basic information

Entry
Database: EMDB / ID: EMD-20945
TitleCryo-EM structure of wild-type bovine multidrug resistance protein 1 (MRP1) under active turnover conditions
Map data3.23-Angstrom cryo-EM reconstruction of wild-type bovine MRP1 under active turnover conditions in the presence of ATP and leukotriene C4
Sample
  • Complex: bovine multidrug resistance protein 1 (MRP1)
    • Protein or peptide: Multidrug resistance-associated protein 1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: CHOLESTEROL
KeywordsABC transporter / multidrug resistance / outward facing / TRANSPORT PROTEIN / MRP1
Function / homology
Function and homology information


Heme degradation / Synthesis of Leukotrienes (LT) and Eoxins (EX) / cyclic nucleotide transport / Transport of RCbl within the body / Paracetamol ADME / leukotriene transport / glutathione transmembrane transporter activity / glutathione transmembrane transport / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity ...Heme degradation / Synthesis of Leukotrienes (LT) and Eoxins (EX) / cyclic nucleotide transport / Transport of RCbl within the body / Paracetamol ADME / leukotriene transport / glutathione transmembrane transporter activity / glutathione transmembrane transport / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / ABC-family proteins mediated transport / Cytoprotection by HMOX1 / ABC-type xenobiotic transporter / ABC-type xenobiotic transporter activity / xenobiotic transport / lipid 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 / ATP-binding cassette, ABC transporter-type domain profile. ...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
Multidrug resistance-associated protein 1
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsJohnson ZL / Chen J
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseJun 10, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uy0
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20945.png

Downloads & links

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Map

FileDownload / File: emd_20945.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3.23-Angstrom cryo-EM reconstruction of wild-type bovine MRP1 under active turnover conditions in the presence of ATP and leukotriene C4
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.09093327 - 0.1840185
Average (Standard dev.)0.000000103241 (±0.0038368243)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z329.600329.600329.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0910.1840.000

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Supplemental data

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Sample components

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Entire : bovine multidrug resistance protein 1 (MRP1)

EntireName: bovine multidrug resistance protein 1 (MRP1)
Components
  • Complex: bovine multidrug resistance protein 1 (MRP1)
    • Protein or peptide: Multidrug resistance-associated protein 1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: CHOLESTEROL

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Supramolecule #1: bovine multidrug resistance protein 1 (MRP1)

SupramoleculeName: bovine multidrug resistance protein 1 (MRP1) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 170 KDa

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Macromolecule #1: Multidrug resistance-associated protein 1

MacromoleculeName: Multidrug resistance-associated protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 172.862562 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MALRDFCSVD GSDLFWEWNV TWNTSNPDFT KCFQNTVLVW VPCSYLWVCF PFYFLYLSHH DRGYIQMTHL NKAKTALGFL LWIVCWADL FYSFWERSMG KLLAPVFLVS PTLLGITMLL ATFLIQIERR RGVQSSGIML TFWLIALLCA LAILRSKIMT A LKEDARVD ...String:
MALRDFCSVD GSDLFWEWNV TWNTSNPDFT KCFQNTVLVW VPCSYLWVCF PFYFLYLSHH DRGYIQMTHL NKAKTALGFL LWIVCWADL FYSFWERSMG KLLAPVFLVS PTLLGITMLL ATFLIQIERR RGVQSSGIML TFWLIALLCA LAILRSKIMT A LKEDARVD VFRDVTFYIY FSLVLIQLVL SCFSDRSPLF SETINDPNPC PESSASFLSR ITFWWITGMM VQGYRQPLES TD LWSLNKE DTSEQVVPVL VKNWKKECAK SRKQPVKIVY SSKDPAKPKG SSKVDVNEEA EALIVKCPQK ERDPSLFKVL YKT FGPYFL MSFLFKAVHD LMMFAGPEIL KLLINFVNDK KAPEWQGYFY TALLFISACL QTLVLHQYFH ICFVSGMRIK TAVI GAVYR KALVITNAAR KSSTVGEIVN LMSVDAQRFM DLATYINMIW SAPLQVILAL YLLWLNLGPS VLAGVAVMVL MVPLN AVMA MKTKTYQVAH MKSKDNRIKL MNEILNGIKV LKLYAWELAF KDKVLAIRQE ELKVLKKSAY LAAVGTFTWV CTPFLV ALS TFAVYVTVDE NNILDAQKAF VSLALFNILR FPLNILPMVI SSIVQASVSL KRLRVFLSHE DLDPDSIQRR PIKDAGA TN SITVKNATFT WARNDPPTLH GITFSVPEGS LVAVVGQVGC GKSSLLSALL AEMDKVEGHV TVKGSVAYVP QQAWIQNI S LRENILFGRQ LQERYYKAVV EACALLPDLE ILPSGDRTEI GEKGVNLSGG QKQRVSLARA VYCDSDVYLL DDPLSAVDA HVGKHIFENV IGPKGLLKNK TRLLVTHAIS YLPQMDVIIV MSGGKISEMG SYQELLARDG AFAEFLRTYA SAEQEQGQPE DGLAGVGGP GKEVKQMENG MLVTDTAGKQ MQRQLSSSSS YSRDVSQHHT STAELRKPGP TEETWKLVEA DKAQTGQVKL S VYWDYMKA IGLFISFLSI FLFLCNHVAS LVSNYWLSLW TDDPIVNGTQ EHTQVRLSVY GALGISQGIT VFGYSMAVSI GG IFASRRL HLDLLHNVLR SPISFFERTP SGNLVNRFSK ELDTVDSMIP QVIKMFMGSL FNVIGACIII LLATPMAAVI IPP LGLIYF FVQRFYVASS RQLKRLESVS RSPVYSHFNE TLLGVSVIRA FEEQERFIRQ SDLKVDENQK AYYPSIVANR WLAV RLECV GNCIVLFASL FAVISRHSLS AGLVGLSVSY SLQVTTYLNW LVRMSSEMET NIVAVERLKE YSETEKEAPW QIQDM APPK DWPQVGRVEF RDYGLRYRED LDLVLKHINV TIDGGEKVGI VGRTGAGKSS LTLGLFRIKE SAEGEIIIDD INIAKI GLH DLRFKITIIP QDPVLFSGSL RMNLDPFSQY SDEEVWTSLE LAHLKGFVSA LPDKLNHECA EGGENLSVGQ RQLVCLA RA LLRKTKILVL DEATAAVDLE TDDLIQSTIR TQFDDCTVLT IAHRLNTIMD YTRVIVLDKG EIQEWGSPSD LLQQRGLF Y SMAKDSGLVS NSLEVLFQ

UniProtKB: Multidrug resistance-associated protein 1

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5.3 mg/mL
BufferpH: 8
Component:
ConcentrationName
150.0 mMKCl
50.0 mMTris
2.0 mMMgCl2
2.0 mMDTT
0.06 %Digitonin
3.0 mMFos-Choline-8, fluorinated
10.0 mMATP-Mg
80.0 uMLeukotriene C4
2.5 %DMSODimethyl sulfoxide
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 3993 / Average exposure time: 10.0 sec. / Average electron dose: 75.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1143729
Startup modelType of model: EMDB MAP
EMDB ID:

7099

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 81078
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6bhu


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-6uy0:
Cryo-EM structure of wild-type bovine multidrug resistance protein 1 (MRP1) under active turnover conditions

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