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- EMDB-50105: Initial 3D Map of relaxosome complex with oriT DNA ds-67_+113(pol... -

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Basic information

Entry
Database: EMDB / ID: EMD-50105
TitleInitial 3D Map of relaxosome complex with oriT DNA ds-67_+113(poly-dT15-17_-3)deltaTraM
Map data3D refinement map Unsharpened
Sample
  • Complex: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM was excluded from complex formation
KeywordsRelaxosome / Bacterial Conjugation / DNA processing / Relaxase / DNA binding proteins / DNA BINDING PROTEIN
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.43 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 15, 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_50105.png

Downloads & links

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Map

FileDownload / File: emd_50105.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D refinement map Unsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 420 pix.
= 347.76 Å		0.83 Å/pix.
x 420 pix.
= 347.76 Å		0.83 Å/pix.
x 420 pix.
= 347.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.2805335 - 0.78170955
Average (Standard dev.)0.0027212382 (±0.027481796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 347.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 7 A low-pass filtered refinement map

Fileemd_50105_additional_1.map
Annotation7 A low-pass filtered refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement map - Half-A

Fileemd_50105_half_map_1.map
AnnotationRefinement map - Half-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement map - Half-B

Fileemd_50105_half_map_2.map
AnnotationRefinement map - Half-B
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 was excluded from complex formation
Components
  • Complex: Complex of the relaxosome containing oriT DNA, accessory protein TraY, host protein IHF and relaxase TraI. TraM was excluded from complex formation

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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 was excluded from complex formation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#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: 50.0 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: 2.4 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: CryoSPARC ab-initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF
Details: A reconstruction resulting from initial processing of the cryoEM dataset to yield a low resolution map showing order/disorder in the trans-esterase (TE) domain of TraI. In this initial 3D ...Details: A reconstruction resulting from initial processing of the cryoEM dataset to yield a low resolution map showing order/disorder in the trans-esterase (TE) domain of TraI. In this initial 3D map of the relaxosome, TE domain is ordered as the oriT DNA (ds-67_+113(poly-dT15-17_-3)deltaTraM) used was conducive to dsDNA melting.
Number images used: 346208
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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