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- PDB-7nb6: Structure of the autoinducer-2 exporter TqsA from E. coli -

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Basic information

Entry
Database: PDB / ID: 7nb6
TitleStructure of the autoinducer-2 exporter TqsA from E. coli
ComponentsAI-2 transport protein TqsA
KeywordsSTRUCTURAL PROTEIN / cryo-EM / membrane protein / autoinducer-2 exporter / quorum sensing / pentamer / protein oligomerization
Function / homologyautoinducer AI-2 transmembrane transport / Transmembrane protein TqsA-like / AI-2E family transporter / quorum sensing / efflux transmembrane transporter activity / plasma membrane => GO:0005886 / transmembrane transport / AI-2 transport protein TqsA
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKhera, R. / Xie, H. / Michel, H.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: EMBO J / Year: 2022
Title: Cryo-EM structures of pentameric autoinducer-2 exporter from Escherichia coli reveal its transport mechanism.
Authors: Radhika Khera / Ahmad R Mehdipour / Jani R Bolla / Joerg Kahnt / Sonja Welsch / Ulrich Ermler / Cornelia Muenke / Carol V Robinson / Gerhard Hummer / Hao Xie / Hartmut Michel /
Abstract: Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for ...Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism.
History
DepositionJan 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: 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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 28, 2022Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AI-2 transport protein TqsA
B: AI-2 transport protein TqsA
C: AI-2 transport protein TqsA
D: AI-2 transport protein TqsA
E: AI-2 transport protein TqsA


Theoretical massNumber of molelcules
Total (without water)187,8185
Polymers187,8185
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "E"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALALEUE1 - 340
d_21ens_1ALALEUB1 - 340
d_31ens_1ALALEUC1 - 340
d_41ens_1ALALEUD1 - 340
d_51ens_1ALALEUA1 - 340

NCS oper:
IDCodeMatrixVector
1given(-0.802343908963, 0.596857923809, 0.0022069290059), (-0.596860125009, -0.802345176244, -0.000457528340442), (0.00149763942684, -0.00168432299948, 0.999997460063)161.174803784, 321.656173495, 0.000911185184322
2given(-0.809863994999, -0.586591610651, -0.00553099608297), (0.586590879944, -0.809881126103, 0.00192383704684), (-0.00560795600808, -0.0016863855028, 0.99998285332)321.648284324, 163.649872116, 0.977359273859
3given(0.317022565491, -0.948406257897, -0.00471836322354), (0.948346788902, 0.316933879257, 0.0138305516603), (-0.0116215725847, -0.00885924158143, 0.999893220744)218.953629848, -37.1648593384, 2.98067341982
4given(0.315792288742, 0.948811297279, -0.0056879281982), (-0.948822439266, 0.31580580123, 0.00163543690107), (0.00334800172969, 0.00488037554531, 0.999982486256)-34.8698140716, 218.552950845, -1.18620769705

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Components

#1: Protein
AI-2 transport protein TqsA / Transport of quorum-sensing signal protein


Mass: 37563.543 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: tqsA, ydgG, b1601, JW1593 / Plasmid: pBADC3 / Production host: Escherichia coli (E. coli) / Strain (production host): Top10 / References: UniProt: P0AFS5

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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: Pentameric TqsA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.187 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pBADC3
Buffer solutionpH: 7.5 / Details: 50 mM Tris (pH 7.5), 150 mM NaCl and 0.006% GDN
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Its a membrane protein and for purification, glyco-diosgenin was used.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: blot time 4 s, blot force +20

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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: OTHER
Electron lensMode: OTHER / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5452
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.5.0particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1218552
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141366 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 109.91 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0113270
ELECTRON MICROSCOPYf_angle_d0.94918130
ELECTRON MICROSCOPYf_dihedral_angle_d25.7511790
ELECTRON MICROSCOPYf_chiral_restr0.052345
ELECTRON MICROSCOPYf_plane_restr0.0052180
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EELECTRON MICROSCOPYNCS constraints0.000700365210545
ens_1d_3EELECTRON MICROSCOPYNCS constraints0.000708454927567
ens_1d_4EELECTRON MICROSCOPYNCS constraints0.0621710737721
ens_1d_5EELECTRON MICROSCOPYNCS constraints0.000702091469195

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