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- EMDB-51370: Full-length MmpL4 in complex with the synthetic nanobody Sb09 and... -

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Basic information

Entry
Database: EMDB / ID: EMD-51370
TitleFull-length MmpL4 in complex with the synthetic nanobody Sb09 and the E. coli acyl carrier protein
Map dataFull-length MmpL4 bound to Sb09 at 6.3 A.
Sample
  • Complex: MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier protein and sybody 09.
KeywordsRND superfamily MmpL family Siderophore export Drug resistance Acyl carrier protein / MEMBRANE PROTEIN
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 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_51370.png

Downloads & links

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Map

FileDownload / File: emd_51370.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull-length MmpL4 bound to Sb09 at 6.3 A.
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.6 Å/pix.
x 100 pix.
= 260. Å		2.6 Å/pix.
x 100 pix.
= 260. Å		2.6 Å/pix.
x 100 pix.
= 260. Å

Surface

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Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.332
Minimum - Maximum-0.44550428 - 1.1341833
Average (Standard dev.)0.0038686323 (±0.051310126)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51370_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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

Fileemd_51370_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_51370_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 : MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier...

EntireName: MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier protein and sybody 09.
Components
  • Complex: MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier protein and sybody 09.

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Supramolecule #1: MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier...

SupramoleculeName: MmpL4 of M. tuberculosis in complex with the E. coli acyl carrier protein and sybody 09.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 129 KDa

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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

Concentration4.2 mg/mL
BufferpH: 7.5 / Details: 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.03% DDM
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

MicroscopeFEI TITAN 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: 6616 / Average exposure time: 1.3 sec. / Average electron dose: 63.7 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: 1540601
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 66189
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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