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- PDB-8ufe: Multidrug efflux pump EfpA from mycobacterium smegmatis -

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Basic information

Entry
Database: PDB / ID: 8ufe
TitleMultidrug efflux pump EfpA from mycobacterium smegmatis
ComponentsIntegral membrane efflux protein EfpA
KeywordsTRANSPORT PROTEIN / multidrug efflux pump / EfpA / Mycobacterium smegmatis
Function / homologyMajor facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / transmembrane transporter activity / MFS transporter superfamily / membrane / PHOSPHATIDYLETHANOLAMINE / Putative MFS-type transporter EfpA
Function and homology information
Biological speciesMycolicibacterium smegmatis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsWang, S. / Liao, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Nat Commun / Year: 2024
Title: Structures of the Mycobacterium tuberculosis efflux pump EfpA reveal the mechanisms of transport and inhibition.
Authors: Shuhui Wang / Kun Wang / Kangkang Song / Zon Weng Lai / Pengfei Li / Dongying Li / Yajie Sun / Ye Mei / Chen Xu / Maofu Liao /
Abstract: As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA ...As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.
History
DepositionOct 4, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Integral membrane efflux protein EfpA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,1342
Polymers48,4001
Non-polymers7341
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Integral membrane efflux protein EfpA


Mass: 48399.910 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Gene: efpA_1 / Production host: Mycolicibacterium smegmatis (bacteria) / References: UniProt: A0A653FLF0
#2: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: multidrug efflux pump MsEfpA / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52150 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033512
ELECTRON MICROSCOPYf_angle_d0.6264788
ELECTRON MICROSCOPYf_dihedral_angle_d7.911528
ELECTRON MICROSCOPYf_chiral_restr0.039595
ELECTRON MICROSCOPYf_plane_restr0.004592

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