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- EMDB-53283: Single particle cryo-EM structure of MdtF V610F with bound Rhodam... -

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Basic information

Entry
Database: EMDB / ID: EMD-53283
TitleSingle particle cryo-EM structure of MdtF V610F with bound Rhodamine 6G
Map data
Sample
  • Complex: R6G-bound homotrimer of V610F multidrug resistance protein MdtF in SMALP nanodisc
    • Protein or peptide: Multidrug resistance protein MdtF
  • Ligand: DODECANE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: RHODAMINE 6G
Keywordsmultidrug / pump / anaerobic / lipid / MEMBRANE PROTEIN
Function / homology
Function and homology information


xenobiotic transmembrane transport / bile acid transmembrane transporter activity / xenobiotic transport / bile acid and bile salt transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / response to toxic substance / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
Hydrophobe/amphiphile efflux-1 HAE1 / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Acriflavin resistance protein / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Multidrug resistance protein MdtF
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLawrence R / Adams C / Sousa JS / Ahdash Z / Reading E
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T008709/1 United Kingdom
CitationJournal: bioRxiv / Year: 2025
Title: Molecular basis for multidrug efflux by an anaerobic RND transporter.
Authors: Ryan Lawrence / Mohd Athar / Muhammad R Uddin / Christopher Adams / Joana S Sousa / Oliver Durrant / Sophie Lellman / Lucy Sutton / C William Keevil / Nisha Patel / Christine Prosser / David ...Authors: Ryan Lawrence / Mohd Athar / Muhammad R Uddin / Christopher Adams / Joana S Sousa / Oliver Durrant / Sophie Lellman / Lucy Sutton / C William Keevil / Nisha Patel / Christine Prosser / David McMillan / Helen I Zgurskaya / Attilio V Vargiu / Zainab Ahdash / Eamonn Reading
Abstract: Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux ...Bacteria can resist antibiotics and toxic substances within demanding ecological settings, such as low oxygen, extreme acid, and during nutrient starvation. MdtEF, a proton motive force-driven efflux pump from the resistance-nodulation-cell division (RND) superfamily, is upregulated in these conditions but its molecular mechanism is unknown. Here, we report cryo-electron microscopy structures of Escherichia coli multidrug transporter MdtF within native-lipid nanodiscs, including a single-point mutant with an altered multidrug phenotype and associated substrate-bound form. We reveal that drug binding domain and channel conformational plasticity likely governs promiscuous substrate specificity, analogous to its closely related, constitutively expressed counterpart, AcrB. Whereas we discover distinct transmembrane state transitions within MdtF, which create a more engaged proton relay network, altered drug transport allostery and an acid-responsive increase in efflux efficiency. Physiologically, this provides means of xenobiotic and metabolite disposal within remodelled cell membranes that presage encounters with acid stresses, as endured in the gastrointestinal tract.
History
DepositionMar 28, 2025-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53283.png

Downloads & links

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Map

FileDownload / File: emd_53283.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 260 pix.
= 246.22 Å		0.95 Å/pix.
x 260 pix.
= 246.22 Å		0.95 Å/pix.
x 260 pix.
= 246.22 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.947 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.19127196 - 0.2823059
Average (Standard dev.)0.00009784581 (±0.010853489)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 246.22 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_53283_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_53283_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : R6G-bound homotrimer of V610F multidrug resistance protein MdtF i...

EntireName: R6G-bound homotrimer of V610F multidrug resistance protein MdtF in SMALP nanodisc
Components
  • Complex: R6G-bound homotrimer of V610F multidrug resistance protein MdtF in SMALP nanodisc
    • Protein or peptide: Multidrug resistance protein MdtF
  • Ligand: DODECANE
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: RHODAMINE 6G

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Supramolecule #1: R6G-bound homotrimer of V610F multidrug resistance protein MdtF i...

SupramoleculeName: R6G-bound homotrimer of V610F multidrug resistance protein MdtF in SMALP nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 334.551 KDa

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Macromolecule #1: Multidrug resistance protein MdtF

MacromoleculeName: Multidrug resistance protein MdtF / type: protein_or_peptide / ID: 1
Details: V610F MdtF protein with a C-terminal 6x His (hexahistidine) tag, separated by a linker sequence including a TEV cleavage site
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 113.807898 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MANYFIDRPV FAWVLAIIMM LAGGLAIMNL PVAQYPQIAP PTITVSATYP GADAQTVEDS VTQVIEQNMN GLDGLMYMSS TSDAAGNAS ITLTFETGTS PDIAQVQVQN KLQLAMPSLP EAVQQQGISV DKSSSNILMV AAFISDNGSL NQYDIADYVA S NIKDPLSR ...String:
MANYFIDRPV FAWVLAIIMM LAGGLAIMNL PVAQYPQIAP PTITVSATYP GADAQTVEDS VTQVIEQNMN GLDGLMYMSS TSDAAGNAS ITLTFETGTS PDIAQVQVQN KLQLAMPSLP EAVQQQGISV DKSSSNILMV AAFISDNGSL NQYDIADYVA S NIKDPLSR TAGVGSVQLF GSEYAMRIWL DPQKLNKYNL VPSDVISQIK VQNNQISGGQ LGGMPQAADQ QLNASIIVQT RL QTPEEFG KILLKVQQDG SQVLLRDVAR VELGAEDYST VARYNGKPAA GIAIKLAAGA NALDTSRAVK EELNRLSAYF PAS LKTVYP YDTTPFIEIS IQEVFKTLVE AIILVFLVMY LFLQNFRATI IPTIAVPVVI LGTFAILSAV GFTINTLTMF GMVL AIGLL VDDAIVVVEN VERVIAEDKL PPKEATHKSM GQIQRALVGI AVVLSAVFMP MAFMSGATGE IYRQFSITLI SSMLL SVFV AMSLTPALCA TILKAAPEGG HKPNALFARF NTLFEKSTQH YTDSTRSLLR CTGRYMVVYL LICAGMAVLF LRTPTS FLP EEDQGVFMTT AQLPSGATMV NTTKVLQQVT DYYLTKEKDN VQSVFTFGGF GFSGQGQNNG LAFISLKPWS ERVGEEN SV TAIIQRAMIA LSSINKAVVF PFNLPAVAEL GTASGFDMEL LDNGNLGHEK LTQARNELLS LAAQSPNQVT GVRPNGLE D TPMFKVNVNA AKAEAMGVAL SDINQTISTA FGSSYVNDFL NQGRVKKVYV QAGTPFRMLP DNINQWYVRN ASGTMAPLS AYSSTEWTYG SPRLERYNGI PSMEILGEAA AGKSTGDAMK FMADLVAKLP AGVGYSWTGL SYQEALSSNQ APALYAISLV VVFLALAAL YESWSIPFSV MLVVPLGVVG ALLATDLRGL SNDVYFQVGL LTTIGLSAKN AILIVEFAVE MMQKEGKTPI E AIIEAARM RLRPILMTSL AFILGVLPLV ISHGAGSGAQ NAVGTGVMGG MFAATVLAIY FVPVFFVVVE HLFARFKKAA SG ENLYFQS LEHHHHHH

UniProtKB: Multidrug resistance protein MdtF

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Macromolecule #2: DODECANE

MacromoleculeName: DODECANE / type: ligand / ID: 2 / Number of copies: 12 / Formula: D12
Molecular weightTheoretical: 170.335 Da
Chemical component information

ChemComp-D12:
DODECANE

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Macromolecule #3: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 3 / Number of copies: 23 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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Macromolecule #4: RHODAMINE 6G

MacromoleculeName: RHODAMINE 6G / type: ligand / ID: 4 / Number of copies: 1 / Formula: RHQ
Molecular weightTheoretical: 443.557 Da
Chemical component information

ChemComp-R6G:
RHODAMINE 6G

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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

Concentration0.5 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 41.25 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000

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Image processing

Particle selectionNumber selected: 4498131
CTF correctionSoftware - Name: CTFFIND (ver. 4.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

24653

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 1967418
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9qps


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9qpt:
Single particle cryo-EM structure of MdtF V610F with bound Rhodamine 6G

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