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- EMDB-40821: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40821
TitleBovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound to ATP in MSP lipid nanodisc
Map datacryoSPARC non-uniform refinement
Sample
  • Organelle or cellular component: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound to ATP in MSP lipid nanodisc
    • Protein or peptide: ATP binding cassette subfamily C member 4
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsABC transporter / multidrug resistance-associated protein / membrane protein / transport protein
Function / homology
Function and homology information


Platelet degranulation / Paracetamol ADME / Azathioprine ADME / ABC-family proteins mediated transport / ATPase-coupled transmembrane transporter activity / ABC-type transporter activity / transmembrane transport / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ATP-binding cassette sub-family C member 4 / : / 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 sub-family C member 4 / : / 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
ATP binding cassette subfamily C member 4
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPourmal S / Stroud RM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM24485 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of prostaglandin efflux by MRP4.
Authors: Sergei Pourmal / Evan Green / Ruchika Bajaj / Ilan E Chemmama / Giselle M Knudsen / Meghna Gupta / Andrej Sali / Yifan Cheng / Charles S Craik / Deanna L Kroetz / Robert M Stroud /
Abstract: Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules ...Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE, PGE and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.
History
DepositionMay 19, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40821.png

Downloads & links

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Map

FileDownload / File: emd_40821.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoSPARC non-uniform refinement
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-1.8004627 - 3.1215327
Average (Standard dev.)0.0033653474 (±0.075139135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Density modification using DeepEmhancer. Inputs are two half...

Fileemd_40821_additional_1.map
AnnotationDensity modification using DeepEmhancer. Inputs are two half maps from cryoSPARC non-uniform refinement, using the "high resolution" protocol.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A of cryoSPARC non-uniform refinement

Fileemd_40821_half_map_1.map
Annotationhalf map A of cryoSPARC non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B of cryoSPARC non-uniform refinement

Fileemd_40821_half_map_2.map
Annotationhalf map B of cryoSPARC non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound ...

EntireName: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound to ATP in MSP lipid nanodisc
Components
  • Organelle or cellular component: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound to ATP in MSP lipid nanodisc
    • Protein or peptide: ATP binding cassette subfamily C member 4
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound ...

SupramoleculeName: Bovine multidrug resistance protein 4 (MRP4) E1202Q mutant bound to ATP in MSP lipid nanodisc
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: ATP binding cassette subfamily C member 4

MacromoleculeName: ATP binding cassette subfamily C member 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 149.586312 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MQPVYPEVKP NPLRNANLCS RIFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGYWDQEVLR AEKDAREPSL TKAIIKCYW KSYVVLGIFT LIEESTRVVQ PIILGKIIGY FENYDPSDSA ALYEAHGYAG VLSACTLVLA ILHHLYFYHV Q CAGMRLRV ...String:
MQPVYPEVKP NPLRNANLCS RIFFWWLNPL FKIGHKRRLE EDDMYSVLPE DRSQHLGEEL QGYWDQEVLR AEKDAREPSL TKAIIKCYW KSYVVLGIFT LIEESTRVVQ PIILGKIIGY FENYDPSDSA ALYEAHGYAG VLSACTLVLA ILHHLYFYHV Q CAGMRLRV AMCHMIYRKA LRLSNSAMGK TTTGQIVNLL SNDVNKFDQV TIFLHFLWAG PLQAIVVTAL LWMEIGISCL AG MAVLIIL LPLQSCIGKL FSSLRSKTAA FTDTRIRTMN EVITGIRIIK MYAWEKSFAD LITNLRRKEI SKILRSSYLR GMN LASFFV ASKIIVFVTF TTYVFLGNVI TASRVFVAVS LYGAVRLTVT LFFPSAVEKV SEAFVSIRRI KNFLLLDEIT QLHS QLPSD GKMIVNVQDF TAFWDKASDT PTLQSLSFTV RPGELLAVVG PVGAGKSSLL SAVLGELPPN QGQVSVHGRI AYVSQ QPWV FSGTVRSNIL FGKKYEKERY EKVIKACALK KDLQLLEDGD LTMIGDRGTT LSGGQKARVN LARAVYQDAD IYLLDD PLS AVDAEVSRHL FELCICQALH EKIRILVTHQ LQYLKAASQI LILKDGQMVQ KGTYTEFLKS GIDFGSLLKK ENEEAEP SP VPGSPTLRNR TFSESSVWSQ QSSRPSLKEA TPEGQDTENI QVTLTEESRS EGKVGFKAYK NYFTAGAHWF IIIFLILV N LAAQVSYILQ DWWLSYWANQ QSALNVTVNG QGNVTEKLDL NWYLGIYSGL TASTVLFGIV RSLLVFFVLV SSSQTLHNQ MFESILRAPV LFFDRNPIGR ILNRFSKDIG HMDDLLPLTY LDFIQTFLQV IGVVGVAVAV IPWIAIPLVP LGIVFFVLRR YFLETSRDV KRLESTTRSP VFSHLSSSLQ GLWTIRAYKA EQRFQELFDS HQDLHSEAWF LFLTTSRWFA VRLDAICAVF V IVVAFGSL ILAKTLDAGQ VGLALSYALT LMGMFQWCVR QSAEVENMMI SVERVIEYTD LEKEAPWEYQ KRPLPSWPHE GV IIFDNVN FSYSLDGPLV LKHLTALIKS KEKVGIVGRT GAGKSSLIAA LFRLSEPEGK IWIDKILTTE IGLHDLRKKM SII PQEPVL FTGTMRKNLD PFNEHSDEEL WNALEEVQLK EAIEDLPGKM DTELAESGSN FSVGQRQLVC LARAILRKNR ILII DQATA NVDPRTDELI QKKIREKFAH CTVLTIAHRL NTIIDSDKIM VLDSGRLKEY DEPYVLLQNR DSLFYKMVQQ LGKAE AAAL TETAKQVYFK RNYPDITHNG HVVMNASSGQ PSAFTIFETA L

UniProtKB: ATP binding cassette subfamily C member 4

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Macromolecule #2: MAGNESIUM ION

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

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCL, 150 mM NaCl, 1 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsMultidrug resistance-associated protein (MRP4) E1202Q mutant in MSP lipid nanodisc

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3176 / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 840205
Startup modelType of model: OTHER / Details: Stochastic gradient descent from particle images
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 104051

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