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- PDB-9s2u: 1:1 complex of M.tuberculosis MmpL5 and M.smegmatis AcpM -

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Basic information

Entry
Database: PDB / ID: 9s2u
Title1:1 complex of M.tuberculosis MmpL5 and M.smegmatis AcpM
Components
  • Meromycolate extension acyl carrier protein
  • 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
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 ...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 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. / Ramakrishan, 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: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural and functional analysis of the MmpS5L5 efflux pump presages 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 multidrug-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 multidrug-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 trimeric architecture distinct from the canonical tripartite RND efflux pumps of gram-negative bacteria. Structure prediction modeling 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.
History
DepositionJul 22, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 8, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Siderophore exporter MmpL5,Green fluorescent protein
I: Meromycolate extension acyl carrier protein


Theoretical massNumber of molelcules
Total (without water)119,9172
Polymers119,9172
Non-polymers00
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 exporter MmpL5,Green fluorescent protein


Mass: 111448.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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.,M. ...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.,M.tuberculosis MmpL5 with deletion of residues 494-687. Fused to a C-terminal GFP-FLAG tag.
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria), (gene. exp.) Aequorea victoria (jellyfish)
Gene: mmpL5, Rv0676c, MTV040.04c, GFP / Plasmid: pMEXC3GF
Production host: Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: P9WJV1, UniProt: P42212
#2: Protein Meromycolate extension acyl carrier protein / ACP


Mass: 8468.430 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: M.smegmatis AcpM
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc2 155 / References: UniProt: A0R0B3
Has protein modificationN

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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: 1:1 complex of M.tuberculosis MmpL5 and M.smegmatis AcpM
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.119 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Buffer solutionpH: 8
Details: 50 mM HEPES pH8.0, 150 mM NaCl, 0.004% LMNG, 50 uM Bedaquiline
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
250 mMHEPES1
30.004 %LMNG1
450 uMBedaquiline1
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. 50 micromolar bedaquiline was added to the sample
Specimen supportDetails: Edwards S150B glow discharger / Grid material: GOLD / 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: 6040
Details: Movies were collected a 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
1cryoSPARC4.6.2particle selection
2EPUimage acquisition
7UCSF ChimeraX1.8model fitting
9cryoSPARC4.6.2initial Euler assignment
10cryoSPARC4.6.2final Euler assignment
12cryoSPARC4.6.23D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4185525
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78900 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 3.2→3.2 Å / Cor.coef. Fo:Fc: 0.811 / SU B: 31.331 / SU ML: 0.544 / ESU R: 0.51
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.4444 --
obs0.4444 66778 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 126.211 Å2
Refinement stepCycle: 1 / Total: 6157
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0126276
ELECTRON MICROSCOPYr_bond_other_d00.0166215
ELECTRON MICROSCOPYr_angle_refined_deg1.2341.7928544
ELECTRON MICROSCOPYr_angle_other_deg0.4271.71714243
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.4495814
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2.024537
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.825101033
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0570.21028
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.027329
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021379
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it14.59611.8163262
ELECTRON MICROSCOPYr_mcbond_other14.59511.8163262
ELECTRON MICROSCOPYr_mcangle_it23.86721.1724074
ELECTRON MICROSCOPYr_mcangle_other23.86421.1754075
ELECTRON MICROSCOPYr_scbond_it13.50613.7183014
ELECTRON MICROSCOPYr_scbond_other13.50413.7193015
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other23.6424.5754471
ELECTRON MICROSCOPYr_long_range_B_refined40.608137.626257
ELECTRON MICROSCOPYr_long_range_B_other40.608137.626258
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.913 4847 -
obs--100 %

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