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- PDB-9f10: CryoEM structure of the F plasmid relaxosome with TraI in its TE ... -

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Basic information

Entry
Database: PDB / ID: 9f10
TitleCryoEM structure of the F plasmid relaxosome with TraI in its TE mode, without accessory protein TraM. Derived from the ss-27_+8ds+9_+143-R_deltaTraM Locally-refined 2.94 A Map.
Components
  • (Integration host factor subunit ...) x 2
  • Multifunctional conjugation protein TraI
  • R-strand DNA (85-MER)
  • Relaxosome protein TraY
  • T-strand DNA (96-MER)
KeywordsDNA BINDING PROTEIN / Relaxosome / Bacterial Conjugation / DNA processing / Relaxase / DNA binding proteins
Function / homology
Function and homology information


IHF-DNA complex / DNA topoisomerase / DNA topoisomerase type I (single strand cut, ATP-independent) activity / DNA-binding transcription activator activity / protein-DNA complex / structural constituent of chromatin / regulation of translation / chromosome / DNA helicase / DNA recombination ...IHF-DNA complex / DNA topoisomerase / DNA topoisomerase type I (single strand cut, ATP-independent) activity / DNA-binding transcription activator activity / protein-DNA complex / structural constituent of chromatin / regulation of translation / chromosome / DNA helicase / DNA recombination / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / DNA replication / transcription cis-regulatory region binding / DNA-templated transcription / regulation of DNA-templated transcription / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Relaxosome protein TraY / TraY domain / DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / TraI, 2B/2B-like domain / TraI, N-terminal subdomain / : / DNA helicase TraI, C-terminal / single-stranded DNA binding TraI N-terminal subdomain / DNA relaxase TraI 2B/2B-like domain ...Relaxosome protein TraY / TraY domain / DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / TraI, 2B/2B-like domain / TraI, N-terminal subdomain / : / DNA helicase TraI, C-terminal / single-stranded DNA binding TraI N-terminal subdomain / DNA relaxase TraI 2B/2B-like domain / TraI helicase-associated ssDNA bd, N-terminal / Integration host factor, alpha subunit / Integration host factor, beta subunit / Conjugative relaxase, N-terminal / TrwC relaxase / TrwC relaxase / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Bacterial DNA-binding protein / bacterial (prokaryotic) histone like domain / Integration host factor (IHF)-like DNA-binding domain superfamily / AAA domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Relaxosome protein TraY / Integration host factor subunit alpha / Integration host factor subunit beta / Multifunctional conjugation protein TraI
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsWilliams, S.M. / Waksman, G.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM Structure of the relaxosome, a complex essential for bacterial mating and the spread of antibiotic resistance genes.
Authors: Sunanda M Williams / Sandra Raffl / Sabine Kienesberger / Aravindan Ilangovan / Ellen L Zechner / Gabriel Waksman /
Abstract: Bacterial mating, or conjugation, was discovered nearly 80 years ago as a process transferring genes from one bacterial cell (the donor) to another (the recipient). It requires three key multiprotein ...Bacterial mating, or conjugation, was discovered nearly 80 years ago as a process transferring genes from one bacterial cell (the donor) to another (the recipient). It requires three key multiprotein complexes in the donor cell: a DNA-processing machinery called the relaxosome, a double-membrane spanning type 4 secretion system (T4SS), and an extracellular appendage termed pilus. While the near-atomic resolution structures of the T4SS and pilus are already known, that of the relaxosome has not been reported to date. Here, we describe the cryo-EM structure of the fully assembled relaxosome encoded by the paradigm F plasmid in two different states corresponding to distinct functional steps along the DNA processing reaction. By varying the structures of model DNAs we delineate conformational changes required to initiate conjugation. Mutational studies of the various protein-protein and protein-DNA interaction hubs suggest a complex sensitive to trigger signals, that could arise from cell-to-cell contacts with recipient cells.
History
DepositionApr 17, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: T-strand DNA (96-MER)
B: R-strand DNA (85-MER)
C: Integration host factor subunit alpha
D: Integration host factor subunit beta
E: Relaxosome protein TraY
F: Relaxosome protein TraY
G: Relaxosome protein TraY
H: Multifunctional conjugation protein TraI
hetero molecules


Theoretical massNumber of molelcules
Total (without water)355,0369
Polymers355,0128
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, cross-linking
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain T-strand DNA (96-MER)


Mass: 52621.707 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#2: DNA chain R-strand DNA (85-MER)


Mass: 41662.676 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)

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Integration host factor subunit ... , 2 types, 2 molecules CD

#3: Protein Integration host factor subunit alpha / IHF-alpha


Mass: 11373.952 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: ihfA, hid, himA, b1712, JW1702 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A6X7
#4: Protein Integration host factor subunit beta / IHF-beta


Mass: 10671.178 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: ihfB, himD, hip, b0912, JW0895 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A6Y1

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Protein , 2 types, 4 molecules EFGH

#5: Protein Relaxosome protein TraY


Mass: 15210.423 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: traY, ECOK12F073 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06627
#6: Protein Multifunctional conjugation protein TraI


Mass: 193050.859 Da / Num. of mol.: 1
Mutation: Y16F single-point mutation, MEFELGT introduced during cloning
Source method: isolated from a genetically manipulated source
Details: TraI in its trans-esterase or relaxase conformation
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: traI, ECOK12F104 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14565, DNA topoisomerase, DNA helicase

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM has been omitted from complex formationCOMPLEX#1-#60MULTIPLE SOURCES
2Heteroduplex OriT DNA containing the nic siteCOMPLEX#1-#21RECOMBINANTPrepared by annealing of chemically synthesised oligos
3IHF heterodimerCOMPLEX#3-#41RECOMBINANT
4Train of three TraY proteinsCOMPLEX#51RECOMBINANT
5Relaxase/helicase protein TraICOMPLEX#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli K-12 (bacteria)83333
33Escherichia coli K-12 (bacteria)83333
44Escherichia coli K-12 (bacteria)83333
55Escherichia coli K-12 (bacteria)83333
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33Escherichia coli BL21(DE3) (bacteria)469008
44Escherichia coli BL21(DE3) (bacteria)469008
55Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5 / Details: 20 mM Hepes pH 7.5, 100 mM NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The complex after assembly and gel filtration was subjected to glutaraldehyde cross-linking
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: OTHER
Electron lensMode: OTHER / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.09 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
Details: Movies were collected in counting mode fractionated over 50 frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7940692
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 330708 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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