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- EMDB-53947: M.tuberculosis MmpS5L5-acpM complex -

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Basic information

Entry
Database: EMDB / ID: EMD-53947
TitleM.tuberculosis MmpS5L5-acpM complex
Map dataUnsharpened cryo-EM map of a Bedaquiline incubated 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,Green fluorescent protein
    • 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 / bioluminescence / generation of precursor metabolites and energy / 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) / Green fluorescent protein, GFP ...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) / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Meromycolate extension acyl carrier protein / Green fluorescent protein / Siderophore export accessory protein MmpS5 / Siderophore exporter MmpL5
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria) / Aequorea victoria (jellyfish) / Mycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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 6, 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_53947.png

Downloads & links

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Map

FileDownload / File: emd_53947.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened cryo-EM map of a Bedaquiline incubated 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: 0.045
Minimum - Maximum-0.09747459 - 0.18995674
Average (Standard dev.)0.0002680565 (±0.0049638045)
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_53947_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened cryo-EM map of Bedaquiline-incubated M.tuberculosis MmpS5L5-acpM complex...

Fileemd_53947_additional_1.map
AnnotationSharpened cryo-EM map of Bedaquiline-incubated 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_53947_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_53947_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,Green fluorescent protein
    • 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
Details: Incubated with 50 micromolar bedaquiline (1.38% DMSO final)
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 412 KDa

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Macromolecule #1: Siderophore export accessory protein MmpS5

MacromoleculeName: Siderophore export accessory protein MmpS5 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 15.348535 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString:
MSIGTLKRAW IPLLILVVVA IAGFTVQRIR TFFGSEGILV TPKVFADDPE PFDPKVVEYE VSGSGSYVNI NYLDLDAKPQ RIDGAALPW SLTLKTTAPS AAPNILAQGD GTSITCRITV DGEVKDERTA TGVDALTYCF VKSA

UniProtKB: Siderophore export accessory protein MmpS5

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Macromolecule #2: Siderophore exporter MmpL5,Green fluorescent protein

MacromoleculeName: Siderophore exporter MmpL5,Green fluorescent protein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 111.448602 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MIVQRTAAPT GSVPPDRHAA RPFIPRMIRT FAVPIILGWL VTIAVLNVTV PQLETVGQIQ AVSMSPDAAP SMISMKHIGK VFEEGDSDS AAMIVLEGQR PLGDAAHAFY DQMIGRLQAD TTHVQSLQDF WGDPLTATGA QSSDGKAAYV QVKLAGNQGE S LANESVEA ...String:
MIVQRTAAPT GSVPPDRHAA RPFIPRMIRT FAVPIILGWL VTIAVLNVTV PQLETVGQIQ AVSMSPDAAP SMISMKHIGK VFEEGDSDS AAMIVLEGQR PLGDAAHAFY DQMIGRLQAD TTHVQSLQDF WGDPLTATGA QSSDGKAAYV QVKLAGNQGE S LANESVEA VKTIVERLAP PPGVKVYVTG SAALVADQQQ AGDRSLQVIE AVTFTVIIVM LLLVYRSIIT SAIMLTMVVL GL LATRGGV AFLGFHRIIG LSTFATNLLV VLAIAAATDY AIFLIGRYQE ARGLGQDRES AYYTMFGGTA HVVLGSGLTI AGA TFCLSF TRLPYFQTLG VPLAIGMVIV VAAALTLGPA IIAVTSRFGK LLEPKRMARV RGWRKVGAAI VRWPGPILVG AVAL ALVGL LTLPGYRTNY NDRNYLPADL PANEGYAAAE RHFSQARMNP EVLMVESDHD MRNSADFLVI NKIAKAIFAV EGISR VQAI TRPDGKPIES FYLPPEVFDN PDFQRGLEQF LSPDGHAVRF IISHEGDPMS QAGIARIAKI KTAAKEAIKG TPLEGS AIY LGGTAAMFKD LSDGNTYDLM IAGISALCLI FIIMLITTRS VVAAAVIVGT VVLSLGASFG LSVLIWQHIL GIELHWL VL AMAVIILLAV GADYNLLLVA RLKEEIHAGI NTGIIRAMGG SGSVVTAAGL VFAFTMMSFA VSELTVMAQV GTTIGMGL L FDTLIVRSFM TPSIAALLGK WFWWPQVVRQ RPIPQPWPSP ASARTFALVA LEVLFQGPQF SKGEELFTGV VPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYITADKQKN GIKANFKIRH NIEDGSVQLA D HYQQNTPI GDGPVLLPDN HYLSTQSALS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK TSDYKDDDDK

UniProtKB: Siderophore exporter MmpL5, Siderophore exporter MmpL5, Green fluorescent protein

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Macromolecule #3: Meromycolate extension acyl carrier protein

MacromoleculeName: Meromycolate extension acyl carrier protein / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: mc2 155
Molecular weightTheoretical: 11.084209 KDa
SequenceString:
MAATQEEIIA GLAEIIEEVT GIEPSEVTPE KSFVDDLDID (4HH)LSMVEIAVQ TEDKYGVKIP DEDLAGLRTV GDVVAY IQK LEEENPEAAA ALREKFAADQ

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
50.0 micromolarBedaquiline
1.38 %DMSO

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 with 50 micromolar Bedaquiline

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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: 6041 / 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

Particle selectionNumber selected: 4900000
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 33185
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-9rgb:
M.tuberculosis MmpS5L5-acpM complex

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