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- EMDB-16816: Structure of TolQR complex from E.coli -

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Basic information

Entry
Database: EMDB / ID: EMD-16816
TitleStructure of TolQR complex from E.coli
Map dataOne of maps used in refinement, Z-flipped for model building.
Sample
  • Complex: TolQ-TolR 5:2 complex
    • Protein or peptide: Tol-Pal system protein TolQ
    • Protein or peptide: Tol-Pal system protein TolR
KeywordsTolQ / TolR / Tol-Pal / complex / inner membrane / MEMBRANE PROTEIN
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 (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWebby MN / Kleanthous C / Press CE
Funding support United Kingdom, European Union, 2 items
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, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16816.png

Downloads & links

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Map

FileDownload / File: emd_16816.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOne of maps used in refinement, Z-flipped for model building.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 249.6 Å		0.83 Å/pix.
x 300 pix.
= 249.6 Å		0.83 Å/pix.
x 300 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.37246636 - 0.6092768
Average (Standard dev.)-0.0007210239 (±0.015531902)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Class one, from 3D classification.

Fileemd_16816_additional_1.map
AnnotationClass one, from 3D classification.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_16816_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_16816_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TolQ-TolR 5:2 complex

EntireName: TolQ-TolR 5:2 complex
Components
  • Complex: TolQ-TolR 5:2 complex
    • Protein or peptide: Tol-Pal system protein TolQ
    • Protein or peptide: Tol-Pal system protein TolR

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Supramolecule #1: TolQ-TolR 5:2 complex

SupramoleculeName: TolQ-TolR 5:2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 170 KDa

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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 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 20.600367 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MARARGRGRR DLKSEINIVP LLDVLLVLLL IFMATAPIIT QSVEVDLPDA TESQAVSSND NPPVIVEVSG IGQYTVVVEK DRLERLPPE QVVAEVSSRF KANPKTVFLI GGAKDVPYDE IIKALNLLHS AGVKSVGLMT QPILEENLYF QGQFGSWSHP Q FEKGGGSG GGSGGGSWSH PQFEKHHHHH H

UniProtKB: Tol-Pal system protein TolR

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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

Concentration7.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 15.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 10 Blot time 3 sec Wait time 2 sec.
Detailssample 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 46.39 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 566486
FSC plot (resolution estimation)

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