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- PDB-9qpr: Single particle cryo-EM structure of the multidrug efflux pump Md... -

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Basic information

Entry
Database: PDB / ID: 9qpr
TitleSingle particle cryo-EM structure of the multidrug efflux pump MdtF from Escherichia coli
ComponentsMultidrug resistance protein MdtF
KeywordsMEMBRANE PROTEIN / multidrug / pump / anaerobic / lipid
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
DODECANE / PHOSPHATIDYLETHANOLAMINE / Multidrug resistance protein MdtF
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsLawrence, R. / Adams, C. / Sousa, J.S. / Ahdash, Z. / Reading, E.
Funding support United Kingdom, 1items
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, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 14, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Multidrug resistance protein MdtF
A: Multidrug resistance protein MdtF
B: Multidrug resistance protein MdtF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,88731
Polymers341,2803
Non-polymers16,60728
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Multidrug resistance protein MdtF


Mass: 113759.867 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: MdtF protein with a C-terminal 6x His (hexahistidine) tag, separated by a linker sequence including a TEV cleavage site
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: mdtF, yhiV, b3514, JW3482 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P37637
#2: Chemical...
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#3: Chemical
ChemComp-D12 / DODECANE


Mass: 170.335 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C12H26
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Homotrimer of WT multidrug resistance protein MdtF in SMALP nanodisc
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.334551 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48.82 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION4particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2501990
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 583735 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: SwissModel / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 39.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004724300
ELECTRON MICROSCOPYf_angle_d0.640232872
ELECTRON MICROSCOPYf_chiral_restr0.04383849
ELECTRON MICROSCOPYf_plane_restr0.00534128
ELECTRON MICROSCOPYf_dihedral_angle_d12.43743765

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