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- EMDB-62251: Cryo-EM structure of inner membrane TolQRA complex in CYMAL-6-Neo... -

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Basic information

Entry
Database: EMDB / ID: EMD-62251
TitleCryo-EM structure of inner membrane TolQRA complex in CYMAL-6-Neopentyl Glycol detergent micelles
Map data
Sample
  • Complex: Inner membrane TolQRA complex in detergent micelles
    • Complex: Pentameric complex of inner membrane Tol-Pal system protein TolQ
      • Protein or peptide: Tol-Pal system protein TolQ
    • Complex: Dimeric complex of inner membrane Tol-Pal system protein TolR
      • Protein or peptide: Tol-Pal system protein TolR
    • Protein or peptide: Tol-Pal system protein TolA
Keywordsbacteria / outer membrane / lipid homeostasis / phospholipid / inner membrane protein / protein complex structure / proton motive force / stator motor / PROTON TRANSPORT
Function / homology
Function and homology information


cellular response to bacteriocin / cell septum assembly / regulation of membrane invagination / bacteriocin transport / toxin transmembrane transporter activity / protein import / virion binding / cell envelope / cell division site / transmembrane transporter activity ...cellular response to bacteriocin / cell septum assembly / regulation of membrane invagination / bacteriocin transport / toxin transmembrane transporter activity / protein import / virion binding / cell envelope / cell division site / transmembrane transporter activity / disordered domain specific binding / protein transport / protein domain specific binding / symbiont entry into host cell / cell division / membrane / plasma membrane
Similarity search - Function
Tol-Pal system, TolQ / Tol-Pal system protein TolR / TolA C-terminal / Tol-Pal system, TolA / : / Biopolymer transport protein ExbD/TolR / Biopolymer transport protein ExbD/TolR / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family
Similarity search - Domain/homology
Tol-Pal system protein TolQ / Tol-Pal system protein TolR / Tol-Pal system protein TolA
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.19 Å
AuthorsYeow J / Chng SS
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Other governmentMOH-000145 Singapore
CitationJournal: J Am Chem Soc / Year: 2025
Title: Structural Insights into the Force-Transducing Mechanism of a Motor-Stator Complex Important for Bacterial Outer Membrane Lipid Homeostasis.
Authors: Jiang Yeow / Chee Geng Chia / Nadege Zi-Lin Lim / Xiaodan Zhao / Jie Yan / Shu-Sin Chng /
Abstract: Gram-negative bacteria assemble an asymmetric outer membrane (OM) that functions as an effective barrier against antibiotics. Building a stable and functional OM requires the assembly and maintenance ...Gram-negative bacteria assemble an asymmetric outer membrane (OM) that functions as an effective barrier against antibiotics. Building a stable and functional OM requires the assembly and maintenance of balanced levels of proteins, lipopolysaccharides, and phospholipids into the bilayer. In , the trans-envelope Tol-Pal complex has recently been established to play a primary role in maintaining OM lipid homeostasis. It is believed that the motor-stator complex TolQR exploits the proton motive force in the inner membrane to induce conformational changes in the TolA effector, ultimately generating a force across the cell envelope to activate processes at the OM. Molecular details of how such force transduction occurs via the TolQRA complex are unknown. Here, we solve structures of the TolQRA complex using single-particle cryo-EM, capturing the transmembrane (TM) regions of the purified complex in two distinct states at ∼3.6 and ∼4.2 Å nominal resolutions. We define how the TolA N-terminal TM helix interacts with an asymmetric TolQR subcomplex in two different positions, revealing how the two TolQRA states are related by rotation of the TolQ pentamer. By considering structural prediction and biochemical evidence for the periplasmic domains of the complex, we propose a working model for how proton passage through the complex induces rotary movement that can be coupled to TolA for force transduction across the cell envelope.
History
DepositionNov 1, 2024-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62251.png

Downloads & links

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Map

FileDownload / File: emd_62251.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 280 pix.
= 233.52 Å		0.83 Å/pix.
x 280 pix.
= 233.52 Å		0.83 Å/pix.
x 280 pix.
= 233.52 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.20339885 - 0.41754556
Average (Standard dev.)0.00041141707 (±0.014164903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 233.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62251_msk_1.map
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Half map: #1

Fileemd_62251_half_map_1.map
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Half map: #2

Fileemd_62251_half_map_2.map
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Sample components

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Entire : Inner membrane TolQRA complex in detergent micelles

EntireName: Inner membrane TolQRA complex in detergent micelles
Components
  • Complex: Inner membrane TolQRA complex in detergent micelles
    • Complex: Pentameric complex of inner membrane Tol-Pal system protein TolQ
      • Protein or peptide: Tol-Pal system protein TolQ
    • Complex: Dimeric complex of inner membrane Tol-Pal system protein TolR
      • Protein or peptide: Tol-Pal system protein TolR
    • Protein or peptide: Tol-Pal system protein TolA

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Supramolecule #1: Inner membrane TolQRA complex in detergent micelles

SupramoleculeName: Inner membrane TolQRA complex in detergent micelles / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Inner membrane complex of pentameric TolQ with dimeric TolR and periplasmic TolA purified in CYMAL-6-Neopentyl Glycol micelles.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Location in cell: inner membrane
Molecular weightTheoretical: 30.8 KDa

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Supramolecule #2: Pentameric complex of inner membrane Tol-Pal system protein TolQ

SupramoleculeName: Pentameric complex of inner membrane Tol-Pal system protein TolQ
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: TolQ
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: Dimeric complex of inner membrane Tol-Pal system protein TolR

SupramoleculeName: Dimeric complex of inner membrane Tol-Pal system protein TolR
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: TolR

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Macromolecule #1: Tol-Pal system protein TolQ

MacromoleculeName: Tol-Pal system protein TolQ / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 25.623662 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTDMNILDLF LKASLLVKLI MLILIGFSIA SWAIIIQRTR ILNAAAREAE AFEDKFWSGI ELSRLYQESQ GKRDNLTGSE QIFYSGFKE FVRLHRANSH APEAVVEGAS RAMRISMNRE LENLETHIPF LGTVGSISPY IGLFGTVWGI MHAFIALGAV K QATLQMVA ...String:
MTDMNILDLF LKASLLVKLI MLILIGFSIA SWAIIIQRTR ILNAAAREAE AFEDKFWSGI ELSRLYQESQ GKRDNLTGSE QIFYSGFKE FVRLHRANSH APEAVVEGAS RAMRISMNRE LENLETHIPF LGTVGSISPY IGLFGTVWGI MHAFIALGAV K QATLQMVA PGIAEALIAT AIGLFAAIPA VMAYNRLNQR VNKLELNYDN FMEEFTAILH RQAFTVSESN KG

UniProtKB: Tol-Pal system protein TolQ

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Macromolecule #2: Tol-Pal system protein TolR

MacromoleculeName: Tol-Pal system protein TolR / type: protein_or_peptide / ID: 2 / Details: N-terminal octa-histidine expression tagged TolR / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 16.746332 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MARARGRGRR DLKSEINIVP LLDVLLVLLL IFMATAPIIT QSVEVDLPDA TESQAVSSND NPPVIVEVSG IGQYTVVVEK DRLERLPPE QVVAEVSSRF KANPKTVFLI GGAKDVPYDE IIKALNLLHS AGVKSVGLMT QPILEHHHHH HHH

UniProtKB: Tol-Pal system protein TolR

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Macromolecule #3: Tol-Pal system protein TolA

MacromoleculeName: Tol-Pal system protein TolA / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 43.232699 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKATEQNDK LKRAIIISAV LHVILFAALI WSSFDENIEA SAGGGGGSSI DAVMVDSGAV VEQYKRMQSQ ESSAKRSDEQ RKMKEQQAA EELREKQAAE QERLKQLEKE RLAAQEQKKQ AEEAAKQAEL KQKQAEEAAA KAAADAKAKA EADAKAAEEA A KKAAADAK ...String:
MSKATEQNDK LKRAIIISAV LHVILFAALI WSSFDENIEA SAGGGGGSSI DAVMVDSGAV VEQYKRMQSQ ESSAKRSDEQ RKMKEQQAA EELREKQAAE QERLKQLEKE RLAAQEQKKQ AEEAAKQAEL KQKQAEEAAA KAAADAKAKA EADAKAAEEA A KKAAADAK KKAEAEAAKA AAEAQKKAEA AAAALKKKAE AAEAAAAEAR KKAATEAAEK AKAEAEKKAA AEKAAADKKA AA EKAAADK KAAEKAAAEK AAADKKAAAE KAAADKKAAA AKAAAEKAAA AKAAAEADDI FGELSSGKNA PKTGGGAKGN NAS PAGSGN TKNNGASGAD INNYAGQIKS AIESKFYDAS SYAGKTCTLR IKLAPDGMLL DIKPEGGDPA LCQAALAAAK LAKI PKPPS QAVYEVFKNA PLDFKP

UniProtKB: Tol-Pal system protein TolA

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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

Concentration4.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMNa2H2PO4/Na2HPO4phosphate-buffered saline
300.0 mMNaClsodium chloride
20.0 mMC3H4N2Imidazole
0.04 mMC47H84O22CYMAL-6 Neopentyl Glycol

Details: Phosphate-buffered saline (PBS) buffer at pH 7.5 (20 mM PBS pH 7.5, 300 mM NaCl) with 20 mM Imidazole and 0.04 mM CYMAL-6 Neopentyl Glycol detergent
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was observed to be monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Tridiem 4K / Energy filter - Slit width: 20 eV
Details: Gatan GIF post-column energy filter operated in zero-loss mode
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: 7469 / Average exposure time: 5.0 sec. / Average electron dose: 56.0 e/Å2
Details: Images were collected in movie-mode at 40 frames per image.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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: 2497487
Details: We performed automated particle picking using Blob and Template Picker for initial template picking and final sampling respectively.
CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.2) / Details: Patch CTF routine in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: Inner membrane protein of Tol-Pal system, TolQRA.
Details: Building of the high-resolution structures of TolQRA was done via approximate fitting of each monomer independently using the Alphafold 3 model of TolQ, TolR(TM), TolA(TM) as rigid bodies ...Details: Building of the high-resolution structures of TolQRA was done via approximate fitting of each monomer independently using the Alphafold 3 model of TolQ, TolR(TM), TolA(TM) as rigid bodies into the density in Chimera, followed by refinement in COOT.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.19 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.2) / Number images used: 54048
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

abu9

chain_id: A, residue_range: 7-224, source_name: AlphaFold, initial_model_type: in silico model

abu9

chain_id: B, residue_range: 8-224, source_name: AlphaFold, initial_model_type: in silico model

abu9

chain_id: C, residue_range: 7-224, source_name: AlphaFold, initial_model_type: in silico model

abu9

chain_id: D, residue_range: 6-224, source_name: AlphaFold, initial_model_type: in silico model

abu9

chain_id: E, residue_range: 7-223, source_name: AlphaFold, initial_model_type: in silico model

abv6

chain_id: F, residue_range: 14-34, source_name: AlphaFold, initial_model_type: in silico model

abv6

chain_id: G, residue_range: 12-34, source_name: AlphaFold, initial_model_type: in silico model

9934

chain_id: H, residue_range: 7-33, source_name: AlphaFold, initial_model_type: in silico model
DetailsBuilding of the high-resolution structures of TolQRA was done via approximate fitting of each monomer independently using the Alphafold 3 model of TolQ, TolR(TM), TolA(TM) as rigid bodies into the density in Chimera, followed by refinement in COOT.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 124.3 / Target criteria: Cross-correlation coefficient
Output model

PDB-9kch:
Cryo-EM structure of inner membrane TolQRA complex in CYMAL-6-Neopentyl Glycol detergent micelles

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