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- PDB-8odt: Structure of TolQR complex from E.coli -

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Basic information

Entry
Database: PDB / ID: 8odt
TitleStructure of TolQR complex from E.coli
Components
  • Tol-Pal system protein TolQ
  • Tol-Pal system protein TolR
KeywordsMEMBRANE PROTEIN / TolQ / TolR / Tol-Pal / complex / inner membrane
Function / homology
Function and homology information


regulation of membrane invagination / cell envelope / bacteriocin transport / protein import / cell division site / transmembrane transporter activity / protein transport / cell cycle / cell division / membrane / plasma membrane
Similarity search - Function
Tol-Pal system, TolQ / Tol-Pal system protein TolR / 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
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWebby, M.N. / Kleanthous, C. / Press, C.E.
Funding support United Kingdom, European Union, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V008056/1 United Kingdom
European Research Council (ERC)742555European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Tunable force transduction through the cell envelope.
Authors: Daniel P Williams-Jones / Melissa N Webby / Cara E Press / Jan M Gradon / Sophie R Armstrong / Joanna Szczepaniak / Colin Kleanthous /
Abstract: The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and ...The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.
History
DepositionMar 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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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


Theoretical massNumber of molelcules
Total (without water)169,3197
Polymers169,3197
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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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 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABU9
#2: Protein Tol-Pal system protein TolR


Mass: 20600.367 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: tolR, b0738, JW0728 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABV6

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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: TolQ-TolR 5:2 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.170 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: sample purified by affinity chromatography (His-tagged TolR) and SEC before application to grids in 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 2 mM EDTA, 0.01% LMNG
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: Blot force 10 Blot time 3 sec Wait time 2 sec

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 46.39 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 566486 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 241.61 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00559476
ELECTRON MICROSCOPYf_angle_d0.676312808
ELECTRON MICROSCOPYf_chiral_restr0.0381478
ELECTRON MICROSCOPYf_plane_restr0.00461627
ELECTRON MICROSCOPYf_dihedral_angle_d12.86493436

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