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- PDB-7ot9: Structure of the AI-2 exporter family protein YdiK from E. coli -

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Basic information

Entry
Database: PDB / ID: 7ot9
TitleStructure of the AI-2 exporter family protein YdiK from E. coli
ComponentsAI-2E member YdiK
KeywordsSTRUCTURAL PROTEIN / cryo-EM / membrane protein / autoinducer-2 exporter / quorum sensing / pentamer / protein oligomerization / AI-2E / transporter
Function / homologyTransmembrane protein TqsA-like / AI-2E family transporter / plasma membrane / Putative transport protein YdiK
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 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
DepositionJun 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AI-2E member YdiK
E: AI-2E member YdiK
D: AI-2E member YdiK
C: AI-2E member YdiK
B: AI-2E member YdiK


Theoretical massNumber of molelcules
Total (without water)199,3295
Polymers199,3295
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, mass spectrometry, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15250 Å2
ΔGint-144 kcal/mol
Surface area60600 Å2

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Components

#1: Protein
AI-2E member YdiK


Mass: 39865.891 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: P0AFS7

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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 YdiK / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.199 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
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.: 3.8 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.
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: 7378
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1crYOLO1.5.0particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1222739
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 619311 / Num. of class averages: 1 / Symmetry type: POINT

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