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- PDB-5n8o: Cryo EM structure of the conjugative relaxase TraI of the F/R1 pl... -

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Basic information

Entry
Database: PDB / ID: 5n8o
TitleCryo EM structure of the conjugative relaxase TraI of the F/R1 plasmid system
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA helicase IHelicase
KeywordsTRANSFERASE / 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 / metabolic process / DNA helicase activity / DNA helicase / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / TraI, 2B/2B-like domain / TraI, N-terminal subdomain / DNA helicase TraI, C-terminal / single-stranded DNA binding TraI N-terminal subdomain / DNA relaxase TraI 2B/2B-like domain / Conjugative relaxase, N-terminal / TrwC relaxase / TrwC relaxase ...DNA helicase, TraI type / Conjugative transfer relaxase protein TraI / TraI, 2B/2B-like domain / TraI, N-terminal subdomain / DNA helicase TraI, C-terminal / single-stranded DNA binding TraI N-terminal subdomain / DNA relaxase TraI 2B/2B-like domain / Conjugative relaxase, N-terminal / TrwC relaxase / TrwC relaxase / AAA domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Multifunctional conjugation protein TraI / DNA helicase I
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsIlangovan, A. / Zanetti, G. / Waksman, G.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
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 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
DepositionFeb 23, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
Revision 1.1May 10, 2017Group: Database references
Revision 1.2May 17, 2017Group: Database references
Revision 1.3May 31, 2017Group: Structure summary
Revision 1.4Jun 28, 2017Group: Database references / Category: citation / Item: _citation.title
Revision 1.5Aug 30, 2017Group: Data collection / Experimental preparation
Category: em_imaging_optics / em_sample_support / em_software
Item: _em_imaging_optics.energyfilter_name / _em_sample_support.grid_type ..._em_imaging_optics.energyfilter_name / _em_sample_support.grid_type / _em_software.fitting_id / _em_software.name / _em_software.version
Revision 1.6Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.7Nov 18, 2020Group: Data collection / Category: em_imaging_optics
Item: _em_imaging_optics.chr_aberration_corrector / _em_imaging_optics.phase_plate / _em_imaging_optics.sph_aberration_corrector

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-3601
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
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')


Theoretical massNumber of molelcules
Total (without water)198,6442
Polymers198,6442
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5120 Å2
ΔGint-48 kcal/mol
Surface area67320 Å2
MethodPISA

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Components

#1: Protein DNA helicase I / Helicase


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
#2: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 6647.284 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1TraI-22mer complexCOMPLEXall0MULTIPLE SOURCES
2TraI proteinCOMPLEX#11RECOMBINANT
322-mer DNA/RNA hybridCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.193 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris1
2100 mMsodium clorideNaClSodium chloride1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot time 4 sec force 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
19PHENIX1.11.1model refinement
Image processingDetails: Frames were aligned and summed with MotionCor2.
CTF correctionDetails: Done within relion software / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 830000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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 buildingSpace: REAL
Details: Please see article for details of model building and refinement
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00811560
ELECTRON MICROSCOPYf_angle_d1.24215708
ELECTRON MICROSCOPYf_dihedral_angle_d11.0489621
ELECTRON MICROSCOPYf_chiral_restr0.0651790
ELECTRON MICROSCOPYf_plane_restr0.0072015

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