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- PDB-9kch: Cryo-EM structure of inner membrane TolQRA complex in CYMAL-6-Neo... -

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Basic information

Entry
Database: PDB / ID: 9kch
TitleCryo-EM structure of inner membrane TolQRA complex in CYMAL-6-Neopentyl Glycol detergent micelles
Components
  • Tol-Pal system protein TolA
  • Tol-Pal system protein TolQ
  • Tol-Pal system protein TolR
KeywordsPROTON TRANSPORT / bacteria / outer membrane / lipid homeostasis / phospholipid / inner membrane protein / protein complex structure / proton motive force / stator motor
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 K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.19 Å
AuthorsYeow, J. / Chng, S.S.
Funding support Singapore, 1items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 9, 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: Tol-Pal system protein TolQ
B: Tol-Pal system protein TolQ
C: Tol-Pal system protein TolQ
D: Tol-Pal system protein TolQ
E: Tol-Pal system protein TolQ
F: Tol-Pal system protein TolR
G: Tol-Pal system protein TolR
H: Tol-Pal system protein TolA


Theoretical massNumber of molelcules
Total (without water)204,8448
Polymers204,8448
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
Tol-Pal system protein TolQ


Mass: 25623.662 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: tolQ, fii, b0737, JW0727 / Plasmid: pET22/42 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABU9
#2: Protein Tol-Pal system protein TolR


Mass: 16746.332 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal octa-histidine expression tagged TolR / Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: tolR, b0738, JW0728 / Plasmid: pET22/42 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABV6
#3: Protein Tol-Pal system protein TolA


Mass: 43232.699 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: tolA, cim, excC, lky, b0739, JW0729 / Plasmid: pET22/42 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P19934
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Inner membrane TolQRA complex in detergent micellesCOMPLEXInner membrane complex of pentameric TolQ with dimeric TolR and periplasmic TolA purified in CYMAL-6-Neopentyl Glycol micelles.all0RECOMBINANT
2Pentameric complex of inner membrane Tol-Pal system protein TolQCOMPLEXTolQ#11RECOMBINANT
3Dimeric complex of inner membrane Tol-Pal system protein TolRCOMPLEXTolR#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.202 MDaNO
210.128 MDaNO
310.0308 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellular location
21Escherichia coli (E. coli)83333K-12inner membrane
32Escherichia coli K-12 (bacteria)83333
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)511693BL21pET22/42
32Escherichia coli BL21(DE3) (bacteria)469008pET22/42
Buffer solutionpH: 7.5
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
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMphosphate-buffered salineNa2H2PO4/Na2HPO41
2300 mMsodium chlorideNaCl1
320 mMImidazoleC3H4N21
40.04 mMCYMAL-6 Neopentyl GlycolC47H84O221
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was observed to be monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7469
Details: Images were collected in movie-mode at 40 frames per image.
EM imaging opticsEnergyfilter name: GIF Tridiem 4K
Details: Gatan GIF post-column energy filter operated in zero-loss mode
Energyfilter slit width: 20 eV
Image scansSampling size: 5.2 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.2particle selection
2SerialEMimage acquisition
4cryoSPARC4.5.2CTF correction
7UCSF Chimera1.15model fitting
8Coot0.8.9model fitting
10cryoSPARC4.5.2initial Euler assignment
11cryoSPARC4.5.2final Euler assignment
12cryoSPARC4.5.2classification
13cryoSPARC4.5.23D reconstruction
14PHENIX1.20.1-4487model refinement
CTF correctionDetails: Patch CTF routine in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2497487
Details: We performed automated particle picking using Blob and Template Picker for initial template picking and final sampling respectively.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54048 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 124.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
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.
Atomic model building

3D fitting-ID: 1 / Source name: AlphaFold / Type: in silico model

IDAccession codeChain-IDChain residue rangeInitial refinement model-ID
1P0ABU9A7-2241
2P0ABU9B8-2241
3P0ABU9C7-2241
4P0ABU9D6-2241
5P0ABU9E7-2231
6P0ABV6F14-342
7P0ABV6G12-342
8P19934H7-333
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049297
ELECTRON MICROSCOPYf_angle_d0.76412574
ELECTRON MICROSCOPYf_dihedral_angle_d4.6481245
ELECTRON MICROSCOPYf_chiral_restr0.0421459
ELECTRON MICROSCOPYf_plane_restr0.0051586

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