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- PDB-7jk8: EmrE S64V mutant bound to tetra(4-fluorophenyl)phosphonium at pH 5.8 -

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Basic information

Entry
Database: PDB / ID: 7jk8
TitleEmrE S64V mutant bound to tetra(4-fluorophenyl)phosphonium at pH 5.8
ComponentsMultidrug SMR transporter
KeywordsMEMBRANE PROTEIN / Small multidrug resistance protein / Antiporter / Drug efflux pump
Function / homology
Function and homology information


EmrE multidrug transporter complex / amino-acid betaine transmembrane transporter activity / choline transmembrane transporter activity / glycine betaine transport / choline transport / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transport / antiporter activity / response to osmotic stress / xenobiotic transmembrane transporter activity ...EmrE multidrug transporter complex / amino-acid betaine transmembrane transporter activity / choline transmembrane transporter activity / glycine betaine transport / choline transport / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transport / antiporter activity / response to osmotic stress / xenobiotic transmembrane transporter activity / transmembrane transporter activity / xenobiotic metabolic process / transmembrane transport / cellular response to xenobiotic stimulus / membrane => GO:0016020 / response to xenobiotic stimulus / DNA damage response / membrane / identical protein binding / plasma membrane
Similarity search - Function
Small drug/metabolite transporter protein family / Small multidrug resistance protein / Small Multidrug Resistance protein
Similarity search - Domain/homology
tetrakis(4-fluorophenyl)phosphanium / Multidrug SMR transporter / Multidrug transporter EmrE
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodSOLID-STATE NMR / molecular dynamics
AuthorsShcherbakov, A.A. / Hisao, G. / Mandala, V.S. / Thomas, N.E. / Soltani, M. / Salter, E.A. / Davis Jr., J.H. / Henzler-Wildman, K.A. / Hong, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM095839 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.
Authors: Shcherbakov, A.A. / Hisao, G. / Mandala, V.S. / Thomas, N.E. / Soltani, M. / Salter, E.A. / Davis Jr., J.H. / Henzler-Wildman, K.A. / Hong, M.
History
DepositionJul 27, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Multidrug SMR transporter
B: Multidrug SMR transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,3623
Polymers23,9512
Non-polymers4111
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: Experiments have been published in previous studies: FRET, cross-linking. Ligand binding was directly probed in this study with NMR heteronuclear dipolar coupling measurements.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3050 Å2
ΔGint-33 kcal/mol
Surface area12540 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 160structures with the least restraint violations
RepresentativeModel #1lowest energy

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Components

#1: Protein Multidrug SMR transporter / Multidrug resistance protein / Multidrug transporter emrE / SMR family multidrug efflux protein EmrE


Mass: 11975.331 Da / Num. of mol.: 2 / Mutation: S64V
Source method: isolated from a genetically manipulated source
Details: Asymmetric homodimer - chemical shift data for two chains are different.
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: emrE, emrE_2, BvCmsNSNP036_03582, D3821_21360, D9G11_17095, EYY27_16395, NCTC12650_04038, SAMEA3752559_04448
Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A2X7QID6, UniProt: P23895*PLUS
#2: Chemical ChemComp-VCJ / tetrakis(4-fluorophenyl)phosphanium


Mass: 411.351 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H16F4P
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
113isotropic13D solid-state hCANH
123isotropic13D solid-state hCONH
133isotropic23D solid-state hCA(CO)NH
143isotropic23D solid-state hCO(CA)NH
152isotropic32D CC CORD
162isotropic33D NCACX
171isotropic11D 19F
181isotropic12D FF Exchange
191isotropic119F-13C Double-Quantum Cross Polarization
1103isotropic12D solid-state hNH
1111isotropic11D 13C-19F REDOR
1123isotropic12D hNH-resolved 1H-19F REDOR

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
membrane10.26 mg/uL [U-99% 13C; U-99% 15N] EmrE-S64V, 9.5 ug/uL F4TPP, 0.41 mg/uL DMPC, 50 mM MES, 20 mM sodium chloride, aqueous bufferU-C,N-labelled S64V EmrE, with F4-TPP in DMPC bilayers at 1:25 P:L ratio.CHN-S64V-EmrE-F4TPP-1:25PLaqueous buffer
membrane20.13 mg/uL [U-99% 13C; U-99% 15N] EmrE-S64V, 5.4 ug/uL F4TPP, 0.46 mg/uL DMPC, 50 mM MES, 20 mM sodium chloride, aqueous bufferU-C,N-labelled S64V EmrE, with F4-TPP in DMPC bilayers at 1:50 P:L ratio.CHN-S64V-EmrE-F4TPP-1:50PLaqueous buffer
membrane30.27 mg/uL [U-99% 13C; U-99% 15N] EmrE-S64V, 9.5 ug/uL F4TPP, 0.44 mg/uL [U-99% 2H] DMPC, 50 mM MES, 20 mM sodium chloride, aqueous bufferU-C,D,N-labelled S64V EmrE, with F4-TPP in d54-DMPC bilayers at 1:25 P:L ratio.CDN-S64V-EmrE-F4TPP-1:25PLaqueous buffer
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.26 mg/uLEmrE-S64V[U-99% 13C; U-99% 15N]1
9.5 ug/uLF4TPPnatural abundance1
0.41 mg/uLDMPCnatural abundance1
50 mMMESnatural abundance1
20 mMsodium chloridenatural abundance1
0.13 mg/uLEmrE-S64V[U-99% 13C; U-99% 15N]2
5.4 ug/uLF4TPPnatural abundance2
0.46 mg/uLDMPCnatural abundance2
50 mMMESnatural abundance2
20 mMsodium chloridenatural abundance2
0.27 mg/uLEmrE-S64V[U-99% 13C; U-99% 15N]3
9.5 ug/uLF4TPPnatural abundance3
0.44 mg/uLDMPC[U-99% 2H]3
50 mMMESnatural abundance3
20 mMsodium chloridenatural abundance3
Sample conditionsIonic strength: 0.07 M / Label: sample_conditions_1 / pH: 5.8 / Pressure: 1 atm / Temperature: 285 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III6001
Bruker AVANCE NEOBrukerAVANCE NEO7002
Bruker AVANCE IIBrukerAVANCE II8003

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Processing

NMR software
NameVersionDeveloperClassification
GROMACS2019.1UNIVERSITY OF GRONINGEN ROYAL INSTITUTE OF TECHNOLOGY UPPSALA UNIVERSITYrefinement
HADDOCK WEBSERVER2.4structure solution
TopSpin2.1-4.0structure solution
NMRFAM-SPARKY1.414structure solution
1.47structure solution
RefinementMethod: molecular dynamics / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 160 / Conformers submitted total number: 10

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