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- PDB-7sfq: EmrE S64V Mutant Bound to tetra(4-fluorophenyl)phosphonium at pH 8.0 -

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Basic information

Entry
Database: PDB / ID: 7sfq
TitleEmrE S64V Mutant Bound to tetra(4-fluorophenyl)phosphonium at pH 8.0
ComponentsMultidrug transporter EmrE
KeywordsMEMBRANE PROTEIN / Multidrug resistance protein / SMR Transporter / Efflux protein / Proton-coupled
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 / 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 transporter EmrE
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodSOLID-STATE NMR / molecular dynamics
AuthorsShcherbakov, A.A. / Spreacker, P.J. / Dregni, A.J. / Henzler-Wildman, K.A. / Hong, M.
Funding support United States, 3items
OrganizationGrant numberCountry
Other private3130800 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM095839 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM088204 United States
CitationJournal: Nat Commun / Year: 2022
Title: High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport.
Authors: Shcherbakov, A.A. / Spreacker, P.J. / Dregni, A.J. / Henzler-Wildman, K.A. / Hong, M.
History
DepositionOct 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


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


  • Idetical with deposited unit
  • defined by author
  • Evidence: fluorescence resonance energy transfer, Experiments have been published in previous studies: FRET, cross-linking.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2920 Å2
ΔGint-34 kcal/mol
Surface area13390 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 82structures with the least restraint violations
RepresentativeModel #1fewest violations

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Components

#1: Protein Multidrug transporter EmrE / Efflux-multidrug resistance protein EmrE / Ethidium resistance protein / Methyl viologen resistance protein C


Mass: 11975.331 Da / Num. of mol.: 2 / Mutation: S64V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: emrE / Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P23895
#2: Chemical ChemComp-VCJ / tetrakis(4-fluorophenyl)phosphanium


Mass: 411.351 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H16F4P / Feature type: SUBJECT OF INVESTIGATION
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
111isotropic12D solid-state hNH
121isotropic13D solid-state hCANH
131isotropic13D solid-state hCONH
141isotropic13D solid-state hCA(CO)NH
151isotropic13D solid-state hCO(CA)NH
191isotropic13D solid-state h(CA)CB(CACO)NH
181isotropic13D solid-state hN(CACO)NH
171isotropic13D solid-state H(NCACO)NH
1101isotropic13D solid-state h(CA)CB(CA)NH
161isotropic12D hNH-resolved 1H-19F REDOR
1111isotropic12D FF Exchange
1121isotropic12D water-edited solid-state hNH

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Sample preparation

DetailsType: liposome
Contents: 0.27 mg/uL [U-13C; U-15N; U-2H] Multidrug Resistance Protein E (EmrE), Complex with tetra(4-fluorophenyl)phosphonium, 9.5 ug/uL F4TPP, 0.44 mg/uL [U-99% 2H] d54-DMPC, bilayers
Details: Uniformly C,D,N-labelled S64V EmrE, with F4-TPP in d54-DMPC bilayers at 1:25 P:L ratio at pH 8.0
Label: Sample_1 / Solvent system: bilayers
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.27 mg/uLMultidrug Resistance Protein E (EmrE), Complex with tetra(4-fluorophenyl)phosphonium[U-13C; U-15N; U-2H]1
9.5 ug/uLF4TPPnatural abundance1
0.44 mg/uLd54-DMPC[U-99% 2H]1
Sample conditionsIonic strength: 0.07 M / Label: Conditions_1 / pH: 8.0 / PH err: 0.1 / Pressure: 1 atm / Temperature: 285 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 600 MHz

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Processing

NMR software
NameVersionDeveloperClassification
HADDOCKWebserver 2.4Alexandre Bonvin and members of the computational structural biology group, Utrecht Universitystructure calculation
GROMACS2019.1UNIVERSITY OF GRONINGEN ROYAL INSTITUTE OF TECHNOLOGY UPPSALA UNIVERSITYrefinement
TopSpin2.1-4.0Bruker Biospincollection
TopSpin2.1-4.0Bruker Biospinprocessing
NMRFAM-SPARKY1.414, 1.470NMRFAMchemical shift assignment
NMRFAM-SPARKY1.414, 1.470NMRFAMpeak picking
RefinementMethod: molecular dynamics / Software ordinal: 2
Details: The authors state that the starting model for structure calculation was 7JK8. The ligand VCJ was removed from the 7JK8 structure and re-docked using HADDOCK, under new constraints measured ...Details: The authors state that the starting model for structure calculation was 7JK8. The ligand VCJ was removed from the 7JK8 structure and re-docked using HADDOCK, under new constraints measured in the present study. The docked structures best agreeing with experiment were further refined by all-atom MD simulations in explicit lipid bilayers to generate the ensemble deposited for this study.
NMR representativeSelection criteria: fewest violations
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 82 / Conformers submitted total number: 10

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