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Yorodumi- PDB-5n8o: Cryo EM structure of the conjugative relaxase TraI of the F/R1 pl... -
+Open data
-Basic information
Entry | Database: PDB / ID: 5n8o | ||||||
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Title | Cryo EM structure of the conjugative relaxase TraI of the F/R1 plasmid system | ||||||
Components |
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Keywords | TRANSFERASE / Relaxase / Cryo EM / Helicase / translocase | ||||||
Function / homology | Function and homology information DNA topoisomerase / DNA topoisomerase type I (single strand cut, ATP-independent) activity / hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / DNA helicase activity / DNA helicase / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Ilangovan, A. / Zanetti, G. / Waksman, G. | ||||||
Funding support | United Kingdom, 1items
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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 trans- ...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. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5n8o.cif.gz | 271.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5n8o.ent.gz | 208.9 KB | Display | PDB format |
PDBx/mmJSON format | 5n8o.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5n8o_validation.pdf.gz | 873.1 KB | Display | wwPDB validaton report |
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Full document | 5n8o_full_validation.pdf.gz | 892.5 KB | Display | |
Data in XML | 5n8o_validation.xml.gz | 47.5 KB | Display | |
Data in CIF | 5n8o_validation.cif.gz | 71.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/n8/5n8o ftp://data.pdbj.org/pub/pdb/validation_reports/n8/5n8o | HTTPS FTP |
-Related structure data
Related structure data | 3601MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 191996.734 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: traI / Production host: Escherichia coli (E. coli) / References: UniProt: Q6TDU5, UniProt: P14565*PLUS |
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#2: DNA chain | Mass: 6647.284 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight | Value: 0.193 MDa / Experimental value: YES | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.2 | ||||||||||||||||||||||||
Buffer component |
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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 K / Details: blot time 4 sec force 1 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | 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 X / Calibrated magnification: 47619 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / 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 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2900 / Details: Total exposure 8 sec for a total dose of 50 e- |
EM imaging optics | Energyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV |
Image scans | Sampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 20 / Used frames/image: 1-20 |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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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 | Num. of particles selected: 830000 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184451 / Algorithm: BACK PROJECTION / Details: Relion automatic procedure / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Space: REAL Details: Please see article for details of model building and refinement | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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