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- PDB-9gi3: Truncated MmpL4 in nanodiscs in the presence of desferrated mycobactin -

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Basic information

Entry
Database: PDB / ID: 9gi3
TitleTruncated MmpL4 in nanodiscs in the presence of desferrated mycobactin
Components
  • Acyl carrier protein
  • Siderophore exporter MmpL4
KeywordsMEMBRANE PROTEIN / RND superfamily MmpL family Siderophore export Drug resistance Acyl carrier protein
Function / homology
Function and homology information


lipid biosynthetic process / lipid A biosynthetic process / acyl binding / acyl carrier activity / phosphopantetheine binding / fatty acid biosynthetic process / response to xenobiotic stimulus / lipid binding / membrane / plasma membrane ...lipid biosynthetic process / lipid A biosynthetic process / acyl binding / acyl carrier activity / phosphopantetheine binding / fatty acid biosynthetic process / response to xenobiotic stimulus / lipid binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Membrane transport protein MmpL family / : / Membrane transport protein MMPL domain / MMPL family / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
: / Acyl carrier protein / Siderophore exporter MmpL4
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Escherichia coli MC1061 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsEarp, J.C. / Garaeva, A.A. / Seeger, M.A.
Funding supportEuropean Union, Switzerland, United States, 4items
OrganizationGrant numberCountry
European Research Council (ERC)772190European Union
University of ZurichFK-21-041 Switzerland
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI151239 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI137338 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of siderophore export and drug efflux by Mycobacterium tuberculosis.
Authors: Jennifer C Earp / Alisa A Garaeva / Virginia Meikle / Michael Niederweis / Markus A Seeger /
Abstract: To replicate and cause disease, Mycobacterium tuberculosis secretes siderophores called mycobactins to scavenge iron from the human host. Two closely related transporters, MmpL4 and MmpL5, are ...To replicate and cause disease, Mycobacterium tuberculosis secretes siderophores called mycobactins to scavenge iron from the human host. Two closely related transporters, MmpL4 and MmpL5, are required for mycobactin secretion and drug efflux. In clinical strains, overproduction of MmpL5 confers resistance towards bedaquiline and clofazimine, key drugs to combat multidrug resistant tuberculosis. Here, we present cryogenic-electron microscopy structures of MmpL4 and identify a mycobactin binding site, which is accessible from the cytosol and also required for bedaquiline efflux. An unusual coiled-coil domain predicted to extend 130 Å into the periplasm is essential for mycobactin and bedaquiline efflux by MmpL4 and MmpL5. The mycobacterial acyl carrier protein MbtL forms a complex with MmpL4, indicating that mycobactin synthesis and export are coupled. Thus, MmpL4 and MmpL5 constitute the core components of a unique multi-subunit machinery required for iron acquisition and drug efflux by M. tuberculosis.
History
DepositionAug 16, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 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: Acyl carrier protein
B: Siderophore exporter MmpL4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,2823
Polymers92,4542
Non-polymers8281
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 Acyl carrier protein / ACP / Cytosolic-activating factor / CAF / Fatty acid synthase acyl carrier protein


Mass: 8645.460 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli MC1061 (bacteria) / References: UniProt: P0A6A8
#2: Protein Siderophore exporter MmpL4


Mass: 83808.508 Da / Num. of mol.: 1 / Mutation: Deletion (S491-Y685)
Source method: isolated from a genetically manipulated source
Details: A coiled-coil domain of MmpL4 (S491-Y685), predicted by AlphaFold2, was deleted and replaced with a short GS linker.
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: mmpL4, MT0466 / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: P9WJV2
#3: Chemical ChemComp-A1IL5 / Mycobactin S / [(2R)-4-oxidanylidene-4-[[(3R)-1-oxidanyl-2-oxidanylidene-azepan-3-yl]amino]butan-2-yl] (2S)-2-[[(4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-oxazol-4-yl]carbonylamino]-6-[octadec-2-enoyl(oxidanyl)amino]hexanoate / [4-[(1-hydroxy-2-oxoazepan-3-yl)amino]-4-oxobutan-2-yl] 6-[hydroxy-[(E)-octadec-2-enoyl]amino]-2-[[2-(2-hydroxyphenyl)-4,5-dihydro-1,3-oxazole-4-carbonyl]amino]hexanoate


Mass: 828.046 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C44H69N5O10 / 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: Complex of truncated MmpL4 from M. tuberculosis bound to the E. coli acyl carrier protein and the substrate desferrated mycobactin.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.1138 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli MC1061 (bacteria)
Buffer solutionpH: 7.5 / Details: 20mM Tris-HCl pH 7.5, 150mM NaCl
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.3 sec. / Electron dose: 64.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8423
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.1.2particle selection
2EPU2.9image acquisition
4cryoSPARC4.1.2CTF correction
7Cootmodel fitting
8ISOLDEmodel fitting
10cryoSPARC4.1.2initial Euler assignment
11cryoSPARC4.1.2final Euler assignment
12cryoSPARC4.1.2classification
13cryoSPARC4.1.23D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2767608
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122605 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 9GI0

9gi0


Accession code: 9GI0 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.5 Å / Cross valid method: NONE
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036220
ELECTRON MICROSCOPYf_angle_d0.5518448
ELECTRON MICROSCOPYf_dihedral_angle_d8.041905
ELECTRON MICROSCOPYf_chiral_restr0.0391006
ELECTRON MICROSCOPYf_plane_restr0.0081071

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