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- EMDB-3601: Cryo EM structure of the conjugative relaxase TraI of the F/R1 pl... -

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Basic information

Entry
Database: EMDB / ID: 3601
TitleCryo EM structure of the conjugative relaxase TraI of the F/R1 plasmid system
Map data
SampleTraI-22mer complex:
TraI protein / 22-mer DNA/RNA hybrid / DNA helicase IHelicase / nucleic-acidNucleic acid
Function / homologyDNA helicase TraI / TrwC relaxase / DNA helicase, TraI type / Conjugative relaxase, N-terminal / Conjugative transfer relaxase protein TraI / TrwC relaxase / P-loop containing nucleoside triphosphate hydrolase / DNA topoisomerase / DNA topoisomerase type I activity / conjugation ...DNA helicase TraI / TrwC relaxase / DNA helicase, TraI type / Conjugative relaxase, N-terminal / Conjugative transfer relaxase protein TraI / TrwC relaxase / P-loop containing nucleoside triphosphate hydrolase / DNA topoisomerase / DNA topoisomerase type I activity / conjugation / DNA helicase activity / DNA helicase / metabolic process / DNA binding / ATP binding / metal ion binding / cytoplasm / Multifunctional conjugation protein TraI / DNA helicase I
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsZanetti G / Ilangovan A / Waksman G
CitationJournal: Cell / Year: 2017
Title: Cryo-EM Structure of a Relaxase Reveals the Molecular Basis of DNA Unwinding during Bacterial Conjugation.
Authors: Aravindan Ilangovan / Christopher W M Kay / Sandro Roier / Hassane El Mkami / Enrico Salvadori / Ellen L Zechner / Giulia Zanetti / Gabriel Waksman
Abstract: Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a ...Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5'-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.
Validation ReportPDB-ID: 5n8o

SummaryFull reportAbout validation report
DateDeposition: Feb 23, 2017 / Header (metadata) release: Apr 5, 2017 / Map release: May 3, 2017 / Last update: Aug 30, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-5n8o
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3601.map.gz (map file in CCP4 format, 28312 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
1.05 Å/pix.
= 201.6 Å
192 pix
1.05 Å/pix.
= 201.6 Å
192 pix
1.05 Å/pix.
= 201.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density
Contour Level:0.065 (by author), 0.065 (movie #1):
Minimum - Maximum-0.14619626 - 0.2710497
Average (Standard dev.)0.0012987428 (0.012253763)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin000
Limit191191191
Spacing192192192
CellA=B=C: 201.59999 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z201.600201.600201.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.1460.2710.001

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Supplemental data

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Sample components

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Entire TraI-22mer complex

EntireName: TraI-22mer complex / Number of components: 5
MassExperimental: 193 kDa

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Component #1: protein, TraI-22mer complex

ProteinName: TraI-22mer complex / Recombinant expression: No
MassExperimental: 193 kDa

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Component #2: protein, TraI protein

ProteinName: TraI protein / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, 22-mer DNA/RNA hybrid

ProteinName: 22-mer DNA/RNA hybrid / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, DNA helicase I

ProteinName: DNA helicase IHelicase / Recombinant expression: No
MassTheoretical: 191.996734 kDa
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: nucleic-acid, DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*...

Nucleic-acidName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)
MassTheoretical: 6.647284 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/ml / pH: 7.2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 % / Details: blot time 4 sec force 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal), 47619 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm / Energy filter: GIF / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2900 / Sampling size: 5 microns / Details: Total exposure 8 sec for a total dose of 50 e-

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 184451 / Details: Frames were aligned and summed with MotionCor2.
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / CTF correction: Done within relion software / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Relion automatic procedure
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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