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- EMDB-53941: M.tuberculosis MmpS5L5-acpM complex -

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Entry
Database: EMDB / ID: EMD-53941
TitleM.tuberculosis MmpS5L5-acpM complex
Map dataUnsharpened cryo-EM map of M.tuberculosis MmpS5L5-acpM complex.
Sample
  • Complex: Trimeric M.tuberculosis MmpS5L5-acpM complex
    • Protein or peptide: Siderophore export accessory protein MmpS5
    • Protein or peptide: Siderophore exporter MmpL5
    • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
KeywordsDrug efflux / RND transporter / Tuberculosis / MEMBRANE PROTEIN
Function / homology
Function and homology information


lipid A biosynthetic process / acyl binding / acyl carrier activity / extracellular region / membrane / plasma membrane / cytosol
Similarity search - Function
Transport accessory protein MmpS / Transport accessory protein MmpS, C-terminal / Mycobacterium membrane protein / Membrane transport protein MmpL family / : / : / Membrane transport protein MMPL domain / MMPL family / Acyl carrier protein (ACP) / Phosphopantetheine attachment site ...Transport accessory protein MmpS / Transport accessory protein MmpS, C-terminal / Mycobacterium membrane protein / Membrane transport protein MmpL family / : / : / Membrane transport protein MMPL domain / MMPL family / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Meromycolate extension acyl carrier protein / Siderophore export accessory protein MmpS5 / Siderophore exporter MmpL5
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria) / Mycobacterium tuberculosis H37Rv (bacteria) / Mycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFountain AJ / Luisi BF / Ramakrishnan L
Funding support United Kingdom, European Union, 3 items
OrganizationGrant numberCountry
Wellcome Trust223103/Z/21/Z United Kingdom
European Research Council (ERC)742210European Union
Wellcome Trust222451/Z/21/Z United Kingdom
CitationJournal: bioRxiv / Year: 2025
Title: Structural and functional analysis of the MmpS5L5 efflux pump presages a pathway to increased bedaquiline resistance.
Authors: Adam J Fountain / Jan Böhning / Stephen H McLaughlin / Tomos E Morgan / Paul H Edelstein / Mark Troll / Meindert H Lamers / Tanmay A M Bharat / Ben F Luisi / Lalita Ramakrishnan
Abstract: Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multi drug resistant tuberculosis. Clinical ...Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multi drug resistant tuberculosis. Clinical bedaquiline resistance in has rapidly emerged, primarily due to mutations in the transcriptional repressor, that result in upregulation of the Resistance-Nodulation-Division (RND) efflux pump MmpS5/MmpL5 (MmpS5L5). Here, to understand how MmpS5L5 effluxes bedaquiline, we determined the structure of the MmpS5L5 complex using cryo-electron microscopy, revealing a novel trimeric architecture distinct from the canonical tripartite RND efflux pumps of Gram-negative bacteria. Structure prediction modelling in conjunction with functional genetic analysis indicates that it uses a periplasmic coiled-coil tube to transport molecules across the cell wall. Structure-guided genetic approaches identify MmpL5 mutations that alter bedaquiline transport; these mutations converge on a region in MmpL5 located in the lower portion of the periplasmic cavity, proximal to the outer leaflet of the inner membrane, suggesting a route for bedaquiline entry into the pump. While currently known clinical resistance to bedaquiline is due to pump upregulation, our findings that several MmpL5 variants increase bedaquiline efflux may presage the emergence of additional modes of clinical resistance.
SIGNIFICANCE STATEMENT: Resistance to bedaquiline, a cornerstone drug for treating multidrug-resistant tuberculosis, is rapidly emerging due to mutations that upregulate expression of the MmpS5L5 ...SIGNIFICANCE STATEMENT: Resistance to bedaquiline, a cornerstone drug for treating multidrug-resistant tuberculosis, is rapidly emerging due to mutations that upregulate expression of the MmpS5L5 efflux pump. Here, we reveal the cryo-EM structure of this pump, showing a novel trimeric architecture and a unique α-helical coiled-coil tube for drug transport. Structure-guided genetic analysis identifies MmpL5 variants that further increase bedaquiline efflux, suggesting potential resistance mechanisms beyond pump upregulation.
History
DepositionJun 5, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53941.png

Downloads & links

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Map

FileDownload / File: emd_53941.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened cryo-EM map of M.tuberculosis MmpS5L5-acpM complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 400 pix.
= 382. Å		0.96 Å/pix.
x 400 pix.
= 382. Å		0.96 Å/pix.
x 400 pix.
= 382. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.955 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.0784335 - 0.16997784
Average (Standard dev.)0.00020318087 (±0.0046085594)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 382.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53941_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened (Cryosparc) cryo-EM map of M.tuberculosis MmpS5L5-acpM complex....

Fileemd_53941_additional_1.map
AnnotationSharpened (Cryosparc) cryo-EM map of M.tuberculosis MmpS5L5-acpM complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_53941_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_53941_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Trimeric M.tuberculosis MmpS5L5-acpM complex

EntireName: Trimeric M.tuberculosis MmpS5L5-acpM complex
Components
  • Complex: Trimeric M.tuberculosis MmpS5L5-acpM complex
    • Protein or peptide: Siderophore export accessory protein MmpS5
    • Protein or peptide: Siderophore exporter MmpL5
    • Protein or peptide: Meromycolate extension acyl carrier protein
  • Ligand: (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: Trimeric M.tuberculosis MmpS5L5-acpM complex

SupramoleculeName: Trimeric M.tuberculosis MmpS5L5-acpM complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 411.9 kDa/nm

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Macromolecule #1: Siderophore export accessory protein MmpS5

MacromoleculeName: Siderophore export accessory protein MmpS5 / type: protein_or_peptide / ID: 1 / Details: M.tuberculosis MmpS5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 3.457247 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString:
SIGTLKRAWI PLLILVVVAI AGFTVQRIRT F

UniProtKB: Siderophore export accessory protein MmpS5

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Macromolecule #2: Siderophore exporter MmpL5

MacromoleculeName: Siderophore exporter MmpL5 / type: protein_or_peptide / ID: 2
Details: M.tuberculosis MmpL5 with deletion of residues 494-687. Fused to a C-terminal GFP-FLAG tag,M.tuberculosis MmpL5 with deletion of residues 494-687. Fused to a C-terminal GFP-FLAG tag
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 79.180367 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: ARPFIPRMIR TFAVPIILGW LVTIAVLNVT VPQLETVGQI QAVSMSPDAA PSMISMKHIG KVFEEGDSDS AAMIVLEGQR PLGDAAHAF YDQMIGRLQA DTTHVQSLQD FWGDPLTATG AQSSDGKAAY VQVKLAGNQG ESLANESVEA VKTIVERLAP P PGVKVYVT ...String:
ARPFIPRMIR TFAVPIILGW LVTIAVLNVT VPQLETVGQI QAVSMSPDAA PSMISMKHIG KVFEEGDSDS AAMIVLEGQR PLGDAAHAF YDQMIGRLQA DTTHVQSLQD FWGDPLTATG AQSSDGKAAY VQVKLAGNQG ESLANESVEA VKTIVERLAP P PGVKVYVT GSAALVADQQ QAGDRSLQVI EAVTFTVIIV MLLLVYRSII TSAIMLTMVV LGLLATRGGV AFLGFHRIIG LS TFATNLL VVLAIAAATD YAIFLIGRYQ EARGLGQDRE SAYYTMFGGT AHVVLGSGLT IAGATFCLSF TRLPYFQTLG VPL AIGMVI VVAAALTLGP AIIAVTSRFG KLLEPKRMAR VRGWRKVGAA IVRWPGPILV GAVALALVGL LTLPGYRTNY NDRN YLPAD LPANEGYAAA ERHFSQARMN PEVLMVESDH DMRNSADFLV INKIAKAIFA VEGISRVQAI TRPDGKPIES FYLPP EVFD NPDFQRGLEQ FLSPDGHAVR FIISHEGDPM SQAGIARIAK IKTAAKEAIK GTPLEGSAIY LGGTAAMFKD LSDGNT YDL MIAGISALCL IFIIMLITTR SVVAAAVIVG TVVLSLGASF GLSVLIWQHI LGIELHWLVL AMAVIILLAV GADYNLL LV ARLKEEIHAG INTGIIRAMG GSGSVVTAAG LVFAFTMMSF AVSELTVMAQ VGTTIGMGLL FDTLIVRSFM TPSIAALL G KWFWWPQVVR QRPIPQPW

UniProtKB: Siderophore exporter MmpL5, Siderophore exporter MmpL5

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Macromolecule #3: Meromycolate extension acyl carrier protein

MacromoleculeName: Meromycolate extension acyl carrier protein / type: protein_or_peptide / ID: 3
Details: M.smegmatis acpM post-translationally modified with a phosphopantethiene group on Ser41.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: mc2 155
Molecular weightTheoretical: 8.808763 KDa
SequenceString:
ATQEEIIAGL AEIIEEVTGI EPSEVTPEKS FVDDLDID(4HH)L SMVEIAVQTE DKYGVKIPDE DLAGLRTVGD VVAYIQ KL

UniProtKB: Meromycolate extension acyl carrier protein

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Macromolecule #4: (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadeca...

MacromoleculeName: (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 4 / Number of copies: 3 / Formula: L9Q
Molecular weightTheoretical: 746.05 Da
Chemical component information

ChemComp-L9Q:
(1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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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

Concentration2.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
50.0 mMHEPES
0.004 %LMNG

Details: 50 mM HEPES pH 8.0, 150 mM NaCl, 0.004% LMNG
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 70 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa / Details: Edwards S150B glow discharger
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis protein preparation contains both monomeric MmpL5-AcpM complexes, and a small subset of trimeric MmpS5L5-AcpM complexes in LMNG.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 6769 / Average exposure time: 6.2 sec. / Average electron dose: 80.0 e/Å2
Details: Images were collected at 0, 20 and 40 degree tilt, approximately equal numbers of movies for each tilt value.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 27426
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: AlphaFold2 prediction of full-length MmpS5-MmpL5 trimeric complex
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9rfu:
M.tuberculosis MmpS5L5-acpM complex

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