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- EMDB-50133: CryoEM map of the F plasmid relaxosome with TraI in its TE mode, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-50133
TitleCryoEM map of the F plasmid relaxosome with TraI in its TE mode, without the accessory protein TraM. ss-27_+8ds+9_+143-R_deltaTraM Global 3.11 A Map.
Map dataF plasmid relaxosome without accessory protein TraM: Global refinement map Unsharpened
Sample
  • Complex: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM has been omitted from complex formation
    • Complex: Heteroduplex OriT DNA containing the nic site
    • Complex: IHF heterodimer
    • Complex: Train of three TraY proteins
    • Complex: Relaxase/helicase protein TraI
KeywordsRelaxosome / Bacterial Conjugation / DNA processing / Relaxase / DNA binding proteins / DNA BINDING PROTEIN
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsWilliams SM / Waksman G
Funding support United Kingdom, 1 items
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 18, 2024-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50133.png

Downloads & links

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Map

FileDownload / File: emd_50133.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationF plasmid relaxosome without accessory protein TraM: Global refinement map Unsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 560 pix.
= 462. Å		0.83 Å/pix.
x 560 pix.
= 462. Å		0.83 Å/pix.
x 560 pix.
= 462. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.3901074 - 1.4147443
Average (Standard dev.)0.0007136897 (±0.02516099)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 462.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Global refinement map - Half-B

Fileemd_50133_half_map_1.map
AnnotationGlobal refinement map - Half-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Global refinement map - Half-A

Fileemd_50133_half_map_2.map
AnnotationGlobal refinement map - Half-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 : Complex of the relaxosome containing oriT DNA, accessory protein ...

EntireName: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM has been omitted from complex formation
Components
  • Complex: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM has been omitted from complex formation
    • Complex: Heteroduplex OriT DNA containing the nic site
    • Complex: IHF heterodimer
    • Complex: Train of three TraY proteins
    • Complex: Relaxase/helicase protein TraI

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Supramolecule #1: Complex of the relaxosome containing oriT DNA, accessory protein ...

SupramoleculeName: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM has been omitted from complex formation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8

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Supramolecule #2: Heteroduplex OriT DNA containing the nic site

SupramoleculeName: Heteroduplex OriT DNA containing the nic site / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Details: Prepared by annealing of chemically synthesised oligos
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: IHF heterodimer

SupramoleculeName: IHF heterodimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #4: Train of three TraY proteins

SupramoleculeName: Train of three TraY proteins / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#7
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Supramolecule #5: Relaxase/helicase protein TraI

SupramoleculeName: Relaxase/helicase protein TraI / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #8
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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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

BufferpH: 7.5 / Details: 20 mM Hepes pH 7.5, 100 mM NaCl
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe complex after assembly and gel filtration was subjected to glutaraldehyde cross-linking

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.09 e/Å2
Details: Movies were collected in counting mode fractionated over 50 frames
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7940692
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: CryoSPARC ab-initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 330708
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: CryoSPARC ab-initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: Homogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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