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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: EMD-3601
TitleCryo EM structure of the conjugative relaxase TraI of the F/R1 plasmid system
Map data
Sample
  • Complex: TraI-22mer complex
    • Complex: TraI protein
      • Protein or peptide: DNA helicase IHelicase
    • Complex: 22-mer DNA/RNA hybrid
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
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
Multifunctional conjugation protein TraI / DNA helicase I
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZanetti G / Ilangovan A / Waksman G
Funding support United Kingdom, 1 items
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, 2017-
Header (metadata) releaseApr 5, 2017-
Map releaseMay 3, 2017-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • 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

emd_3601.png

Downloads & links

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Map

FileDownload / File: emd_3601.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.14619626 - 0.2710497
Average (Standard dev.)0.0012987428 (±0.012253763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: TraI-22mer complex
    • Complex: TraI protein
      • Protein or peptide: DNA helicase IHelicase
    • Complex: 22-mer DNA/RNA hybrid
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

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Supramolecule #1: TraI-22mer complex

SupramoleculeName: TraI-22mer complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 193 KDa

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Supramolecule #2: TraI protein

SupramoleculeName: TraI protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: 22-mer DNA/RNA hybrid

SupramoleculeName: 22-mer DNA/RNA hybrid / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: DNA helicase I

MacromoleculeName: DNA helicase I / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 191.996734 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMSIAQVRSA GSAGNYYTDK DNYYVLGSMG ERWAGKGAEQ LGLQGSVDKD VFTRLLEGRL PDGADLSRMQ DGSNKHRPGY DLTFSAPKS VSMMAMLGGD KRLIDAHNQA VDFAVRQVEA LASTRVMTDG QSETVLTGNL VMALFNHDTS RDQEPQLHTH A VVANVTQH ...String:
MMSIAQVRSA GSAGNYYTDK DNYYVLGSMG ERWAGKGAEQ LGLQGSVDKD VFTRLLEGRL PDGADLSRMQ DGSNKHRPGY DLTFSAPKS VSMMAMLGGD KRLIDAHNQA VDFAVRQVEA LASTRVMTDG QSETVLTGNL VMALFNHDTS RDQEPQLHTH A VVANVTQH NGEWKTLSSD KVGKTGFIEN VYANQIAFGR LYREKLKEQV EALGYETEVV GKHGMWEMPG VPVEAFSGRS QA IREAVGE DASLKSRDVA ALDTRKSKQH VDPEIRMAEW MQTLKETGFD IRAYRDAADQ RTEIRTQAPG PASQDGPDVQ QAV TQAIAG LSERKVQFTY TDVLARTVGI LPPENGVIER ARAGIDEAIS REQLIPLDRE KGLFTSGIHV LDELSVRALS RDIM KQNRV TVHPEKSVPR TAGYSDAVSV LAQDRPSLAI VSGQGGAAGQ RERVAELVMM AREQGREVQI IAADRRSQMN LKQDE RLSG ELITGRRQLL EGMAFTPGST VIVDQGEKLS LKETLTLLDG AARHNVQVLI TDSGQRTGTG SALMAMKDAG VNTYRW QGG EQRPATIISE PDRNVRYARL AGDFAASVKA GEESVAQVSG VREQAILTQA IRSELKTQGV LGHPEVTMTA LSPVWLD SR SRYLRDMYRP GMVMEQWNPE TRSHDRYVID RVTAQSHSLT LRDAQGETQV VRISSLDSSW SLFRPEKMPV ADGERLRV T GKIPGLRVSG GDRLQVASVS EDAMTVVVPG RAEPASLPVS DSPFTALKLE NGWVETPGHS VSDSATVFAS VTQMAMDNA TLNGLARSGR DVRLYSSLDE TRTAEKLARH PSFTVVSEQI KARAGETLLE TAISLQKAGL HTPAQQAIHL ALPVLESKNL AFSMVDLLT EAKSFAAEGT GFTELGGEIN AQIKRGDLLY VDVAKGYGTG LLVSRASYEA EKSILRHILE GKEAVTPLME R VPGELMET LTSGQRAATR MILETSDRFT VVQGYAGVGK TTQFRAVMSA VNMLPASERP RVVGLGPTHR AVGEMRSAGV DA QTLASFL HDTQLQQRSG ETPDFSNTLF LLDESSMVGN TEMARAYALI AAGGGRAVAS GDTDQLQAIA PGQSFRLQQT RSA ADVVIM KEIVRQTPEL REAVYSLINR DVERALSGLE SVKPSQVPRL EGAWAPEHSV TEFSHSQEAK LAEAQQKAML KGEA FPDIP MTLYEAIVRD YTGRTPEARE QTLIVTHLNE DRRVLNSMIH DAREKAGELG KEQVMVPVLN TANIRDGELR RLSTW EKNP DALALVDNVY HRIAGISKDD GLITLQDAEG NTRLISPREA VAEGVTLYTP DKIRVGTGDR MRFTKSDRER GYVANS VWT VTAVSGDSVT LSDGQQTRVI RPGQERAEQH IDLAYAITAH GAQGASETFA IALEGTEGNR KLMAGFESAY VALSRMK QH VQVYTDNRQG WTDAINNAVQ KGTAHDVLEP KPDREVMNAQ RLFSTARELR DVAAGRAVLR QAGLAGGDSP ARFIAPGR K YPQPYVALPA FDRNGKSAGI WLNPLTTDDG NGLRGFSGEG RVKGSGDAQF VALQGSRNGE SLLADNMQDG VRIARDNPD SGVVVRIAGE GRPWNPGAIT GGRVWGDIPD NSVQPGAGNG EPVTAEVLAQ RQAEEAIRRE TERRADEIVR KMAENKPDLP DGKTELAVR DIAGQERDRS AISERETALP ESVLRESQRE REAVREVARE NLLQERLQQM ERDMVRDLQK EKTLGGD

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

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 6.647284 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.2
Component:
ConcentrationNameFormula
50.0 mMtris
100.0 mMsodium clorideNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III / Details: blot time 4 sec force 1.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 47619 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2900 / Average exposure time: 0.4 sec. / Average electron dose: 2.5 e/Å2 / Details: Total exposure 8 sec for a total dose of 50 e-
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 830000
CTF correctionSoftware - Name: CTFFIND (ver. 4) / Details: Done within relion software
Startup modelType of model: INSILICO MODEL / In silico model: EMAN2 starting model generator
Details: 10 models were generated from selected 2D classes, and used in relion 3D refinement. 2 models converged to the same structure, at ~10A resolution, and this structure was used further.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0) / Details: Automatic relion procedures
Final 3D classificationSoftware - Name: RELION (ver. 2.0) / Details: Please see article for details of processing
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Details: Relion automatic procedure / Number images used: 184451
DetailsFrames were aligned and summed with MotionCor2.
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsPlease see article for details of model building and refinement
RefinementSpace: REAL
Output model

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

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