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- PDB-8wm5: multidrug transporter EfpA from Mycobacterium tuberculosis bound ... -

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Basic information

Entry
Database: PDB / ID: 8wm5
Titlemultidrug transporter EfpA from Mycobacterium tuberculosis bound with lipids
ComponentsEfflux pump A
KeywordsTRANSPORT PROTEIN / multidrug transporter / EfpA / mycobacterium / lipid
Function / homology
Function and homology information


transmembrane transporter activity / membrane / plasma 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
CARDIOLIPIN / PHOSPHATIDYLETHANOLAMINE / Uncharacterized MFS-type transporter EfpA
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsWang, S. / Liao, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) 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 3, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Efflux pump A
B: Efflux pump A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,6407
Polymers111,2402
Non-polymers4,4005
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11TRPTRPGLNGLNAA45 - 51945 - 519
d_12PTYPTYPTYPTYAC601
d_13PTYPTYPTYPTYAD602
d_21TRPTRPGLNGLNBB45 - 51945 - 519
d_22PTYPTYPTYPTYBE601
d_23PTYPTYPTYPTYBF602

NCS oper: (Code: givenMatrix: (-0.999999381859, 0.00107001729769, -0.000302231941025), (-0.00107097300276, -0.999994370619, 0.0031798974998), (-0.000298827694317, 0.00318021921642, 0.999994898441) ...NCS oper: (Code: given
Matrix: (-0.999999381859, 0.00107001729769, -0.000302231941025), (-0.00107097300276, -0.999994370619, 0.0031798974998), (-0.000298827694317, 0.00318021921642, 0.999994898441)
Vector: 278.312443065, 278.106353234, -0.464870667185)

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Components

#1: Protein Efflux pump A


Mass: 55620.098 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: efpA / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P9WJY5
#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-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C81H156O17P2 / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: complex of multidrug efflux pump EfpA with lipids / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: OTHER / 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49783 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 37.65 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00887316
ELECTRON MICROSCOPYf_angle_d0.89039924
ELECTRON MICROSCOPYf_chiral_restr0.03961196
ELECTRON MICROSCOPYf_plane_restr0.00691206
ELECTRON MICROSCOPYf_dihedral_angle_d17.5712575
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.103023936357 Å

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