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- EMDB-51369: Truncated MmpL4 in nanodiscs in the presence of desferrated mycobactin -

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Basic information

Entry
Database: EMDB / ID: EMD-51369
TitleTruncated MmpL4 in nanodiscs in the presence of desferrated mycobactin
Map dataTruncated MmpL4 in nanodiscs bound to desferrated mycobactin and the E. coli acyl carrier protein at 3.5 A sharpened at -89.2 A^2.
Sample
  • Complex: Complex of truncated MmpL4 from M. tuberculosis bound to the E. coli acyl carrier protein and the substrate desferrated mycobactin.
    • Protein or peptide: Acyl carrier protein
    • Protein or peptide: Siderophore exporter MmpL4
  • Ligand: Mycobactin S
KeywordsRND superfamily MmpL family Siderophore export Drug resistance Acyl carrier protein / MEMBRANE 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsEarp JC / Garaeva AA / Seeger MA
Funding supportEuropean Union, Switzerland, United States, 4 items
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, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51369.png

Downloads & links

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Map

FileDownload / File: emd_51369.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTruncated MmpL4 in nanodiscs bound to desferrated mycobactin and the E. coli acyl carrier protein at 3.5 A sharpened at -89.2 A^2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 200 pix.
= 260. Å		1.3 Å/pix.
x 200 pix.
= 260. Å		1.3 Å/pix.
x 200 pix.
= 260. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.36
Minimum - Maximum-33.061084999999999 - 45.588709999999999
Average (Standard dev.)-0.000000000009267 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51369_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 2 used for post processing step and...

Fileemd_51369_half_map_1.map
Annotationhalf-map 2 used for post processing step and FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1 used for post processing step and...

Fileemd_51369_half_map_2.map
Annotationhalf-map 1 used for post processing step and FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of truncated MmpL4 from M. tuberculosis bound to the E. c...

EntireName: Complex of truncated MmpL4 from M. tuberculosis bound to the E. coli acyl carrier protein and the substrate desferrated mycobactin.
Components
  • Complex: Complex of truncated MmpL4 from M. tuberculosis bound to the E. coli acyl carrier protein and the substrate desferrated mycobactin.
    • Protein or peptide: Acyl carrier protein
    • Protein or peptide: Siderophore exporter MmpL4
  • Ligand: Mycobactin S

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Supramolecule #1: Complex of truncated MmpL4 from M. tuberculosis bound to the E. c...

SupramoleculeName: Complex of truncated MmpL4 from M. tuberculosis bound to the E. coli acyl carrier protein and the substrate desferrated mycobactin.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 113.8 KDa

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Macromolecule #1: Acyl carrier protein

MacromoleculeName: Acyl carrier protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli MC1061 (bacteria)
Molecular weightTheoretical: 8.64546 KDa
SequenceString:
MSTIEERVKK IIGEQLGVKQ EEVTNNASFV EDLGADSLDT VELVMALEEE FDTEIPDEEA EKITTVQAAI DYINGHQA

UniProtKB: Acyl carrier protein

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Macromolecule #2: Siderophore exporter MmpL4

MacromoleculeName: Siderophore exporter MmpL4 / type: protein_or_peptide / ID: 2
Details: A coiled-coil domain of MmpL4 (S491-Y685), predicted by AlphaFold2, was deleted and replaced with a short GS linker.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 83.808508 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: VSTKFANDSN TNARPEKPFI ARMIHAFAVP IILGWLAVCV VVTVFVPSLE AVGQERSVSL SPKDAPSFEA MGRIGMVFKE GDSDSFAMV IIEGNQPLGD AAHKYYDGLV AQLRADKKHV QSVQDLWGDP LTAAGVQSND GKAAYVQLSL AGNQGTPLAN E SVEAVRSI ...String:
VSTKFANDSN TNARPEKPFI ARMIHAFAVP IILGWLAVCV VVTVFVPSLE AVGQERSVSL SPKDAPSFEA MGRIGMVFKE GDSDSFAMV IIEGNQPLGD AAHKYYDGLV AQLRADKKHV QSVQDLWGDP LTAAGVQSND GKAAYVQLSL AGNQGTPLAN E SVEAVRSI VESTPAPPGI KAYVTGPSAL AADMHHSGDR SMARITMVTV AVIFIMLLLV YRSIITVVLL LITVGVELTA AR GVVAVLG HSGAIGLTTF AVSLLTSLAI AAGTDYGIFI IGRYQEARQA GEDKEAAYYT MYRGTAHVIL GSGLTIAGAT FCL SFARMP YFQTLGIPCA VGMLVAVAVA LTLGPAVLHV GSRFGLFDPK RLLKVRGWRR VGTVVVRWPL PVLVATCAIA LVGL LALPG YKTSYNDRDY LPDFIPANQG YAAADRHFSQ ARMKPEILMI ESDHDMRNPA DFLVLDKLAK GIFRVPGISR VQAIT RPEG TTMDHTGGSS SPPEVFKNKD FQRAMKSFLS SDGHAARFII LHRGDPQSPE GIKSIDAIRT AAEESLKGTP LEDAKI YLA GTAAVFHDIS EGAQWDLLIA AISSLCLIFI IMLIITRAFI AAAVIVGTVA LSLGASFGLS VLLWQHILAI HLHWLVL AM SVIVLLAVGS DYNLLLVSRF KQEIGAGLKT GIIRSMGGTG KVVTNAGLVF AVTMASMAVS DLRVIGQVGT TIGLGLLF D TLIVRSFMTP SIAALLGRWF WWPLRVRSRP ARTPTVPSET QPAGRPLAMS SDRLGALEVL FQ

UniProtKB: Siderophore exporter MmpL4, Siderophore exporter MmpL4

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Macromolecule #3: Mycobactin S

MacromoleculeName: Mycobactin S / type: ligand / ID: 3 / Number of copies: 1 / Formula: A1IL5
Molecular weightTheoretical: 828.046 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.7 mg/mL
BufferpH: 7.5 / Details: 20mM Tris-HCl pH 7.5, 150mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 39.0 kPa / Details: at 15 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 8423 / Average exposure time: 1.3 sec. / Average electron dose: 64.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2767608
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 122605
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9gi0


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

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

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