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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
DescriptorDNA helicase I/DNA Complex
KeywordsTRANSFERASE / Relaxase / Cryo EM / Helicase / translocase / Transferase
Specimen sourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
MethodElectron microscopy (3.9 Å resolution / Particle / Single particle)
AuthorsIlangovan, A. / Zanetti, G. / Waksman, G.
CitationCell, 2017, 169, 708-721.e12

Cell, 2017, 169, 708-721.e12 StrPapers
Cryo-EM Structure of a Relaxase Reveals the Molecular Basis of DNA Unwinding during Bacterial Conjugation.
Aravindan Ilangovan / Christopher W M Kay / Sandro Roier / Hassane El Mkami / Enrico Salvadori / Ellen L Zechner / Giulia Zanetti / Gabriel Waksman

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 23, 2017 / Release: May 3, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 3, 2017Structure modelrepositoryInitial release
1.1May 10, 2017Structure modelDatabase references
1.2May 17, 2017Structure modelDatabase references
1.3May 31, 2017Structure modelStructure summary
1.4Jun 28, 2017Structure modelDatabase referencescitation_citation.title
1.5Aug 30, 2017Structure modelData collection / Experimental preparationem_imaging_optics / em_sample_support / em_software_em_imaging_optics.energyfilter_name / _em_sample_support.grid_type / _em_software.fitting_id / _em_software.name / _em_software.version

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Structure visualization

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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
Polyers198,6442
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)5120
ΔGint (kcal/M)-48
Surface area (Å2)67320
MethodPISA

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Components

#1: Polypeptide(L)DNA helicase I


Mass: 191996.734 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: Q6TDU5

Molecular function

Biological process

#2: DNA chainDNA (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: (synth.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1TraI-22mer complexCOMPLEX1, 20MULTIPLE SOURCES
2TraI proteinCOMPLEX11RECOMBINANT
322-mer DNA/RNA hybridCOMPLEX21RECOMBINANT
Molecular weightValue: 0.193 deg. / Units: MEGADALTONS / Experimental value: YES
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
12562Escherichia coli
23562Escherichia coli
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
12562Escherichia coli
2332630synthetic construct
Buffer solutionpH: 7.2
Buffer component
IDConc.UnitsNameFormulaBuffer ID
150mMtris1
2100mMsodium clorideNaCl1
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 kelvins / Details: blot time 4 sec force 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV / Chr aberration corrector: none / Phase plate: none / Sph aberration corrector: none
Image scansSampling size: 5 microns / Dimension width: 3838 / Dimension 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
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1GautomatchPARTICLE SELECTION1
2EPUIMAGE ACQUISITION1
4CTFFIND4CTF CORRECTION1
7UCSF ChimeraMODEL FITTING1
9RELION2.0INITIAL EULER ASSIGNMENT1
10RELION2.0FINAL EULER ASSIGNMENT1
11RELION2.0CLASSIFICATION1
12RELION2.0RECONSTRUCTION1
19PHENIX1.11.1MODEL REFINEMENT1
Image processingDetails: Frames were aligned and summed with MotionCor2.
CTF correctionDetails: Done within relion software / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 830000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 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 buildingDetails: Please see article for details of model building and refinement
Ref space: REAL
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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