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- PDB-9bii: Mycobacterium tuberculosis EFPA antiparallel dimer -

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Basic information

Entry
Database: PDB / ID: 9bii
TitleMycobacterium tuberculosis EFPA antiparallel dimer
ComponentsUncharacterized MFS-type transporter EfpA
KeywordsMEMBRANE PROTEIN / transporter
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
: / CHOLESTEROL HEMISUCCINATE / Uncharacterized MFS-type transporter EfpA
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKhandelwal, N.K. / Gupta, M. / Stroud, R.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM024485 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A.
Authors: Nitesh Kumar Khandelwal / Meghna Gupta / James E Gomez / Sulyman Barkho / Ziqiang Guan / Ashley Y Eng / Tomohiko Kawate / Sree Ganesh Balasubramani / Andrej Sali / Deborah T Hung / Robert M Stroud /
Abstract: A broad chemical genetic screen in Mycobacterium tuberculosis (Mtb) identified compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of ...A broad chemical genetic screen in Mycobacterium tuberculosis (Mtb) identified compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition, we determined the structures of EfpA with these inhibitors bound at 2.7-3.4 Å resolution. Our structures reveal different mechanisms of inhibition by the two inhibitors. BRD-8000.3 binds in a tunnel contacting the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the movement necessary for alternate access of the transporter. Our results show EfpA as a potential lipid transporter, explain the basis of the synergy of these inhibitors and their potential for combination anti-tuberculosis therapy.
History
DepositionApr 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Uncharacterized MFS-type transporter EfpA
B: Uncharacterized MFS-type transporter EfpA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,68410
Polymers133,2042
Non-polymers5,4808
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 Uncharacterized MFS-type transporter EfpA / Efflux protein A


Mass: 66602.242 Da / Num. of mol.: 2 / Mutation: P171R
Source method: isolated from a genetically manipulated source
Details: Amino acid 1-4 start codon and GSSG linker, Amnio acid 5- 12 Flag tag, Amnio acid 13-15 SGS linker, Amino acid 16-21 thrombin site, Amino acid 22-128 BRIL-Tag, Amino acid 129-610 ...Details: Amino acid 1-4 start codon and GSSG linker, Amnio acid 5- 12 Flag tag, Amnio acid 13-15 SGS linker, Amino acid 16-21 thrombin site, Amino acid 22-128 BRIL-Tag, Amino acid 129-610 Mycobacterium tuberculosis EfpA (truncated from 48 N terminal amino acid) with mutation of Proline at position 171 to Arginine, Amino acid 611-612 SS linker, Amino acid 613-619 TEV site, Amino acid 620-629 10XHis.
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: efpA, Rv2846c / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P9WJY5
#2: Chemical
ChemComp-A1H2V / [(2~{R})-3-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-2-hexadecanoyloxy-propyl] octadecanoate / (2R,3S,4R,5R,6R)-6-((1R,2R,3S,4R,6S)-4,6-DIAMINO-2,3-DIHYDROXYCYCLOHEXYLOXY)-5-AMINO-2-(AMINOMETHYL)-TETRAHYDRO-2H-PYRA N-3,4-DIOL / NEOMYCIN A / NEAMINE / (1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2,6-diamino-2,6-dideoxy-alpha-D-glucoside / (1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2,6-diamino-2,6-dideoxy-D-glucoside / (1R,2R,3S,4R,6S)-4,6-diamino-2,3-dihydroxycyclohexyl 2,6-diamino-2,6-dideoxy-glucoside


Mass: 751.023 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C40H79O10P
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: EfpA / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.06653956 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: H37Rv
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.5 / Details: 50mM Tris-HCL, 300 mM NaCL, 0.02% GDN, 0.002 %CHS
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMSodium chlorideNaCl1
250 mMTrisTris1
SpecimenConc.: 16.16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 second hold 30 second glow / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K

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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: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16938

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.2.1particle selection
2SerialEMv4.1 beta softwareimage acquisition
4cryoSPARCv4.2.1CTF correction
9PHENIXmodel refinement
11cryoSPARCv4.2.1final Euler assignment
12cryoSPARCv4.2.1classification
13cryoSPARCv4.2.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 10225285
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256203 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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