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

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Basic information

Entry
Database: PDB / ID: 9rgb
TitleM.tuberculosis MmpS5L5-acpM complex
Components
  • Meromycolate extension acyl carrier protein
  • Siderophore export accessory protein MmpS5
  • Siderophore exporter MmpL5,Green fluorescent protein
KeywordsMEMBRANE PROTEIN / Drug efflux / RND transporter / Tuberculosis
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
Chem-L9Q / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFountain, A.J. / Luisi, B.F. / Ramakrishnan, L.
Funding support United Kingdom, European Union, 3items
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, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 13, 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: Siderophore export accessory protein MmpS5
B: Siderophore export accessory protein MmpS5
C: Siderophore export accessory protein MmpS5
D: Siderophore exporter MmpL5,Green fluorescent protein
E: Siderophore exporter MmpL5,Green fluorescent protein
F: Siderophore exporter MmpL5,Green fluorescent protein
G: Meromycolate extension acyl carrier protein
H: Meromycolate extension acyl carrier protein
I: Meromycolate extension acyl carrier protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,88212
Polymers413,6449
Non-polymers2,2383
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 Siderophore export accessory protein MmpS5


Mass: 15348.535 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: mmpS5, Rv0677c, MTV040.05c / Plasmid: pMEXC3GF
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain (production host): mc2 155 / References: UniProt: P9WJS7
#2: Protein Siderophore exporter MmpL5,Green fluorescent protein


Mass: 111448.602 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria), (gene. exp.) Aequorea victoria (jellyfish)
Gene: mmpL5, Rv0676c, MTV040.04c, GFP
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain (production host): mc2 155 / References: UniProt: P9WJV1, UniProt: P42212
#3: Protein Meromycolate extension acyl carrier protein / ACP


Mass: 11084.209 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc2 155 / References: UniProt: A0R0B3
#4: Chemical ChemComp-L9Q / (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine


Mass: 746.050 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C41H80NO8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: Trimeric M.tuberculosis MmpS5L5-acpM complex / Type: COMPLEX
Details: Incubated with 50 micromolar bedaquiline (1.38% DMSO final)
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.412 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Plasmid: pMEXC3GF
Buffer solutionpH: 8 / Details: 50 mM HEPES pH 8.0, 150 mM NaCl, 0.004% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
250 mMHEPES1
30.004 %LMNG1
450 micromolarBedaquiline1
51.38 %DMSO1
SpecimenConc.: 2.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This protein preparation contains both monomeric MmpL5-AcpM complexes, and a small subset of trimeric MmpS5L5-AcpM complexes in LMNG with 50 micromolar Bedaquiline
Specimen supportDetails: Edwards S150B glow discharger / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.2 sec. / Electron dose: 80 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6041
Details: Images were collected at 0, 20 and 40 degree tilt, approximately equal numbers of movies for each tilt value.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
7UCSF ChimeraX1.8model fitting
8PHENIX1.21.2_5419model fitting
10PHENIX1.21.2_5419model refinement
11cryoSPARC4.6.2initial Euler assignment
12cryoSPARC4.6.2final Euler assignment
14cryoSPARC4.6.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4900000
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33185 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: AlphaFold2 prediction of full-length MmpS5-MmpL5 trimeric complex
Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00519773
ELECTRON MICROSCOPYf_angle_d0.90326881
ELECTRON MICROSCOPYf_dihedral_angle_d7.8982851
ELECTRON MICROSCOPYf_chiral_restr0.0523227
ELECTRON MICROSCOPYf_plane_restr0.0073377

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