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- EMDB-55353: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuber... -

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Basic information

Entry
Database: EMDB / ID: EMD-55353
TitleAsymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain
Map dataCryo-EM map of the hexameric RND transporter MmpL4-MmpS4 in DDM at a 3.22 A resolution. Map was sharpened with a b factor of 63.9 A.
Sample
  • Complex: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain
    • Complex: MmpL4
      • Protein or peptide: Siderophore exporter MmpL4
    • Complex: MmpS4
      • Protein or peptide: Siderophore export accessory protein MmpS4
  • Ligand: DODECYL-BETA-D-MALTOSIDE
KeywordsRND superfamily / MmpL family / siderophore export / drug resistance / MEMBRANE PROTEIN
Function / homology
Function and homology information


peptidoglycan-based cell wall / extracellular region / plasma membrane
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
Similarity search - Domain/homology
Siderophore export accessory protein MmpS4 / Siderophore exporter MmpL4
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsLichti NP / Earp JC / Garaeva AA / Seeger MA
Funding supportEuropean Union, Switzerland, United States, Germany, 5 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 Al151239 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 Al137338 United States
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: bioRxiv / Year: 2026
Title: Structural elucidation of the hexameric MmpS4-MmpL4 complex from .
Authors: Jennifer C Earp / Nicolas P Lichti / Alisa A Garaeva / Virginia Meikle / Michael Niederweis / Markus A Seeger /
Abstract: contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners ... contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)-(MmpL4) complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60° relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4's conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.
History
DepositionOct 14, 2025-
Header (metadata) releaseJan 28, 2026-
Map releaseJan 28, 2026-
UpdateJan 28, 2026-
Current statusJan 28, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55353.png

Downloads & links

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Map

FileDownload / File: emd_55353.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the hexameric RND transporter MmpL4-MmpS4 in DDM at a 3.22 A resolution. Map was sharpened with a b factor of 63.9 A.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 300 pix.
= 390. Å		1.3 Å/pix.
x 300 pix.
= 390. Å		1.3 Å/pix.
x 300 pix.
= 390. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.8806909 - 1.7374879
Average (Standard dev.)0.00071705115 (±0.028606053)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 390.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_55353_msk_1.map
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_55353_half_map_1.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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Half map: Half-map 2 used for post processing step and...

Fileemd_55353_half_map_2.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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Sample components

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Entire : Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuber...

EntireName: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain
Components
  • Complex: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain
    • Complex: MmpL4
      • Protein or peptide: Siderophore exporter MmpL4
    • Complex: MmpS4
      • Protein or peptide: Siderophore export accessory protein MmpS4
  • Ligand: DODECYL-BETA-D-MALTOSIDE

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Supramolecule #1: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuber...

SupramoleculeName: Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 15 KDa

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Supramolecule #2: MmpL4

SupramoleculeName: MmpL4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Cysteine-depleted (C39S, C319S, C335S, C393S, C780S) full-length MmpL4 with S434C mutation
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Supramolecule #3: MmpS4

SupramoleculeName: MmpS4 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: Full-length MmpS4 with D39C mutation
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: Siderophore exporter MmpL4

MacromoleculeName: Siderophore exporter MmpL4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 105.31707 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: VSTKFANDSN TNARPEKPFI ARMIHAFAVP IILGWLAVSV VVTVFVPSLE AVGQERSVSL SPKDAPSFEA MGRIGMVFKE GDSDSFAMV IIEGNQPLGD AAHKYYDGLV AQLRADKKHV QSVQDLWGDP LTAAGVQSND GKAAYVQLSL AGNQGTPLAN E SVEAVRSI ...String:
VSTKFANDSN TNARPEKPFI ARMIHAFAVP IILGWLAVSV VVTVFVPSLE AVGQERSVSL SPKDAPSFEA MGRIGMVFKE GDSDSFAMV IIEGNQPLGD AAHKYYDGLV AQLRADKKHV QSVQDLWGDP LTAAGVQSND GKAAYVQLSL AGNQGTPLAN E SVEAVRSI VESTPAPPGI KAYVTGPSAL AADMHHSGDR SMARITMVTV AVIFIMLLLV YRSIITVVLL LITVGVELTA AR GVVAVLG HSGAIGLTTF AVSLLTSLAI AAGTDYGIFI IGRYQEARQA GEDKEAAYYT MYRGTAHVIL GSGLTIAGAT FSL SFARMP YFQTLGIPSA VGMLVAVAVA LTLGPAVLHV GSRFGLFDPK RLLKVRGWRR VGTVVVRWPL PVLVATSAIA LVGL LALPG YKTSYNDRDY LPDFIPANQG YAAADRHFCQ ARMKPEILMI ESDHDMRNPA DFLVLDKLAK GIFRVPGISR VQAIT RPEG TTMDHTSIPF QISMQNAGQL QTIKYQRDRA NDMLKQADEM ATTIAVLTRM HSLMAEMAST THRMVGDTEE MKEITE ELR DHVADFDDFW RPIRSYFYWE KHCYGIPICW SFRSIFDALD GIDKLSEQIG VLLGDLREMD RLMPQMVAQI PPQIEAM EN MRTMILTMHS TMTGIFDQML EMSDNATAMG KAFDAAKNDD SFYLPPEVFK NKDFQRAMKS FLSSDGHAAR FIILHRGD P QSPEGIKSID AIRTAAEESL KGTPLEDAKI YLAGTAAVFH DISEGAQWDL LIAAISSLSL IFIIMLIITR AFIAAAVIV GTVALSLGAS FGLSVLLWQH ILAIHLHWLV LAMSVIVLLA VGSDYNLLLV SRFKQEIGAG LKTGIIRSMG GTGKVVTNAG LVFAVTMAS MAVSDLRVIG QVGTTIGLGL LFDTLIVRSF MTPSIAALLG RWFWWPLRVR SRPARTPTVP SETQPAGRPL A MSSDRLGA

UniProtKB: Siderophore exporter MmpL4

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Macromolecule #2: Siderophore export accessory protein MmpS4

MacromoleculeName: Siderophore export accessory protein MmpS4 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 15.493614 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
MSLMRTWIPL VILVVVIVGG FTVHRIRGFF GSENRPSYSC TNLENSKPFN PKHLTYEIFG PPGTVADISY FDVNSEPQRV DGAVLPWSL HITTNDAAVM GNIVAQGNSD SIGCRITVDG KVRAERVSNE VNAYTYCLVK SA

UniProtKB: Siderophore export accessory protein MmpS4

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Macromolecule #3: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 3 / Number of copies: 3 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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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

Concentration4.0 mg/mL
BufferpH: 7.5 / Details: 20mM Tris-HC1 pH 7.5, 150mM NaC1, 0.03% DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / 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

MicroscopeTFS 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: 14584 / Average exposure time: 4.0 sec. / Average electron dose: 62.92 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 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: 9066275
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.0) / Number images used: 76903
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

9gi0

residue_range: 1-783, source_name: PDB, initial_model_type: experimental modelentire monomer used

1-v6

residue_range: 487-690, source_name: AlphaFold, initial_model_type: in silico modelcoiled coil domain used
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 63.9
Output model

PDB-9syt:
Asymmetric hexameric MmpS4-MmpL4 complex from Mycobacterium tuberculosis with "long" coiled-coil domain

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