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- PDB-9e2v: Cryo-EM map of homodecameric TraT -

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Basic information

Entry
Database: PDB / ID: 9e2v
TitleCryo-EM map of homodecameric TraT
ComponentsTraT complement resistance protein
KeywordsMEMBRANE PROTEIN / Membrane protein involved in surface exclusion
Function / homologyEnterobacterial TraT complement resistance / Enterobacterial TraT complement resistance protein / cell outer membrane / Prokaryotic membrane lipoprotein lipid attachment site profile. / TraT complement resistance protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 Å
AuthorsLundgren, C.A.K. / Lea, S. / Deme, J.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Commun Biol / Year: 2025
Title: Structure of the conjugation surface exclusion protein TraT.
Authors: Nicolas Chen / Alfredas Bukys / Camilla A K Lundgren / Justin C Deme / Hafez El Sayyed / Achillefs N Kapanidis / Susan M Lea / Ben C Berks /
Abstract: Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In ...Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In incompatibility group F (IncF) plasmids surface exclusion is mediated by the outer membrane protein TraT. Here we report the cryoEM structure of the TraT exclusion protein complex from the canonical F plasmid of Escherichia coli. TraT is a hollow homodecamer shaped like a chef's hat. In contrast to most outer membrane proteins, TraT spans the outer membrane using transmembrane α-helices. We develop a microscopy-based conjugation assay to probe the effects of directed mutagenesis on TraT. Our analysis provides no support for the idea that TraT has specific interactions with partner proteins. Instead, we infer that TraT is most likely to function by physical interference with conjugation. This work provides structural insight into a natural inhibitor of microbial gene transfer.
History
DepositionOct 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 16, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Apr 16, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TraT complement resistance protein
B: TraT complement resistance protein
C: TraT complement resistance protein
D: TraT complement resistance protein
E: TraT complement resistance protein
F: TraT complement resistance protein
G: TraT complement resistance protein
H: TraT complement resistance protein
I: TraT complement resistance protein
J: TraT complement resistance protein


Theoretical massNumber of molelcules
Total (without water)276,68110
Polymers276,68110
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, C-terminally TS tagged TraT was purified using affinity chromatography and run over a gel filtration column. The sample eluted with a retention volume indicating a higher ...Evidence: gel filtration, C-terminally TS tagged TraT was purified using affinity chromatography and run over a gel filtration column. The sample eluted with a retention volume indicating a higher order assembly, which was verified by single particle cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "H"
d_2ens_1chain "B"
d_3ens_1chain "D"
d_4ens_1chain "A"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "C"
d_9ens_1chain "I"
d_10ens_1chain "J"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: CYS / Beg label comp-ID: CYS / End auth comp-ID: ILE / End label comp-ID: ILE / Auth seq-ID: 22 - 247 / Label seq-ID: 1 - 226

Dom-IDAuth asym-IDLabel asym-ID
d_1HH
d_2BB
d_3DD
d_4AA
d_5EE
d_6FF
d_7GG
d_8CC
d_9II
d_10JJ

NCS oper:
IDCodeMatrixVector
1given(-0.809096688915, -0.587675435405, 0.0003613994542), (0.587675278723, -0.809096754306, -0.00045710951125), (0.000561039156453, -0.000157460267046, 0.999999830221)449.059151023, 228.975866653, -0.0976903555649
2given(-0.808892696827, 0.587956245537, 0.000241565953347), (-0.587956266956, -0.808892717013, -2.25925593215E-5), (0.000182117503985, -0.000160305172391, 0.999999970568)228.757083095, 449.158683888, -0.016880622538
3given(-0.3028341417, -0.953042277539, -0.0013783487051), (0.953042056517, -0.302835750018, 0.00116061222183), (-0.00152352577912, -0.000962151278459, 0.999998376566)422.975154785, 65.4967748148, 0.427700482646
4given(-0.308964293688, 0.951073576363, 0.000342885280702), (-0.951073568237, -0.308964389148, 0.000272101896071), (0.000364728264731, -0.000242039357282, 0.999999904195)66.9993292285, 423.45448522, -0.0424883948877
5given(0.309099047134, 0.95102981544, 0.000263067900526), (-0.951029851006, 0.309099023569, 0.000126979970924), (3.94477071272E-5, -0.000289434814259, 0.999999957336)-48.7974606575, 307.664021681, 0.027044116142
6given(0.809016320248, 0.587785441594, -0.00093178419812), (-0.587786077963, 0.80901609782, -0.000692834862982), (0.000346590170084, 0.00110820449071, 0.999999325879)-74.1682195725, 146.06757293, -0.250269088523
7given(-0.999993139476, 0.00344938885513, 0.00135008062732), (-0.00344887627138, -0.999993979717, 0.000381813165939), (0.00135138952153, 0.000377154285461, 0.99999901575)373.982681915, 375.380162597, -0.336239853301
8given(0.808915145962, -0.587925300737, -0.000356912722651), (0.587925218645, 0.808915223505, -0.000313787744751), (0.000473195889015, 4.39896688442E-5, 0.999999887075)146.049710187, -74.3011644184, -0.115280617242
9given(0.323495430074, -0.946229601877, 0.000497245620126), (0.946229676122, 0.323495547573, 0.000175291429073), (-0.000326722683305, 0.000413802585848, 0.99999986101)304.037678956, -50.0432876126, 0.0134674698552

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Components

#1: Protein
TraT complement resistance protein


Mass: 27668.051 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: traT, ECOK12F101 / Production host: Escherichia coli (E. coli) / References: UniProt: P13979
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

ComponentName: TraT / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 100 mM Tris-HCl, 150 mM NaCl, 0.02% DDM
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTris-HCl1
2150 mMsodium chlorideNaCl1
30.02 %DDM1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / 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: 277.15 K

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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: 800 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: dev_5246 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90771 / Symmetry type: POINT
Atomic model buildingB value: 74.55 / Target criteria: CC
Atomic model buildingAccession code: P13979 / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.55 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002217090
ELECTRON MICROSCOPYf_angle_d0.352723090
ELECTRON MICROSCOPYf_chiral_restr0.0392720
ELECTRON MICROSCOPYf_plane_restr0.00162970
ELECTRON MICROSCOPYf_dihedral_angle_d3.38292390
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2HHELECTRON MICROSCOPYNCS constraints7.25827247945E-13
ens_1d_3HHELECTRON MICROSCOPYNCS constraints3.41296270802E-12
ens_1d_4HHELECTRON MICROSCOPYNCS constraints2.94536671407E-12
ens_1d_5HHELECTRON MICROSCOPYNCS constraints3.57856216204E-12
ens_1d_6HHELECTRON MICROSCOPYNCS constraints6.59728110829E-13
ens_1d_7HHELECTRON MICROSCOPYNCS constraints7.93679151406E-13
ens_1d_8HHELECTRON MICROSCOPYNCS constraints7.01598224636E-13
ens_1d_9HHELECTRON MICROSCOPYNCS constraints6.04173583348E-13
ens_1d_10HHELECTRON MICROSCOPYNCS constraints1.89039082399E-12

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