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- PDB-8ufd: Multidrug efflux pump MtEfpA bound with inhibitor BRD8000.3 -

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Basic information

Entry
Database: PDB / ID: 8ufd
TitleMultidrug efflux pump MtEfpA bound with inhibitor BRD8000.3
ComponentsIntegral membrane efflux protein EFPA
KeywordsTRANSPORT PROTEIN / multidrug efflux pump / EfpA / mycobacterium / BRD8000.3
Function / homology
Function and homology information


transmembrane transporter activity / membrane
Similarity search - Function
Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
PHOSPHATIDYLETHANOLAMINE / : / MFS-type transporter EfpA
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 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

Downloads & links

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Assembly

Deposited unit
A: Integral membrane efflux protein EFPA
B: Integral membrane efflux protein EFPA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,9838
Polymers111,2402
Non-polymers3,7436
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: 55620.098 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: efpA / Production host: Homo sapiens (human) / References: UniProt: A0A045GQW7
#2: Chemical
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C40H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Chemical ChemComp-WJI / (1S,3S)-N-[6-bromo-5-(pyrimidin-2-yl)pyridin-2-yl]-2,2-dimethyl-3-(2-methylpropyl)cyclopropane-1-carboxamide


Mass: 403.316 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H23BrN4O / Feature type: SUBJECT OF INVESTIGATION
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: Complex of Multidrug efflux pump MtEfpA with lipids and inhibitor BRD8000.3
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Homo sapiens (human)
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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 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: 1.19.2_4158: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52099 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037306
ELECTRON MICROSCOPYf_angle_d0.869928
ELECTRON MICROSCOPYf_dihedral_angle_d7.8051134
ELECTRON MICROSCOPYf_chiral_restr0.041198
ELECTRON MICROSCOPYf_plane_restr0.0051210

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