|Entry||Database: PDB / ID: 5n8o|
|Title||Cryo EM structure of the conjugative relaxase TraI of the F/R1 plasmid system|
|Keywords||TRANSFERASE / Relaxase / Cryo EM / Helicase / translocase / Transferase|
|Function/homology||DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / DNA helicase TraI / AAA domain / Conjugative relaxase, N-terminal / TrwC relaxase / TrwC relaxase / conjugation / DNA topoisomerase / DNA topoisomerase type I activity ...DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / DNA helicase TraI / AAA domain / Conjugative relaxase, N-terminal / TrwC relaxase / TrwC relaxase / conjugation / DNA topoisomerase / DNA topoisomerase type I activity / DNA helicase activity / DNA helicase / metabolic process / P-loop containing nucleoside triphosphate hydrolase / DNA binding / ATP binding / metal ion binding / cytoplasm / Multifunctional conjugation protein TraI / DNA helicase I|
Function and homology information
|Specimen source||Escherichia coli / / bacteria /|
|Method||Electron microscopy (3.9 Å resolution / Particle / Single particle) / Transmission electron microscopy|
|Authors||Ilangovan, A. / Zanetti, G. / Waksman, G.|
|Citation||Journal: 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.
Copyright: 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
SummaryFull reportAbout validation report
|Date||Deposition: Feb 23, 2017 / Release: May 3, 2017|
Downloads & links
A: DNA helicase I
C: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
|#1: Protein/peptide|| |
Mass: 191996.734 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / / bacteria / / Gene: traI / Production host: Escherichia coli / References: UniProt:Q6TDU5, UniProt:P14565*PLUS
|#2: DNA chain|| |
Mass: 6647.284 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli / / bacteria /
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE|
|Molecular weight||Value: 0.193 deg. / Units: MEGADALTONS / Experimental value: YES|
|Buffer solution||pH: 7.2|
|Specimen||Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: GOLD / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 kelvins / Details: blot time 4 sec force 1|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD / Nominal magnification: 130000 / Calibrated magnification: 47619 / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 0.4 sec. / Electron dose: 2.5 e/Å2 / Details: Total exposure 8 sec for a total dose of 50 e- / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 2900|
|EM imaging optics||Energyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV / Chr aberration corrector: none / Phase plate: none / Sph aberration corrector: none|
|Image scans||Sampling size: 5 microns / Dimension width: 3838 / Dimension height: 3710 / Movie frames/image: 20 / Used frames/image: 1-20|
|Software||Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement|
|Image processing||Details: Frames were aligned and summed with MotionCor2.|
|CTF correction||Details: Done within relion software / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Number of particles selected: 830000|
|Symmetry||Point symmetry: C1|
|3D reconstruction||Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 184451 / Algorithm: BACK PROJECTION / Details: Relion automatic procedure / Number of class averages: 1 / Symmetry type: POINT|
|Atomic model building||Details: Please see article for details of model building and refinement|
Ref space: REAL
|Refine LS restraints|
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