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- PDB-9d5l: The C-terminal domain of Thiopseudomonas alkaliphila Tn7 TnsE bou... -

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Basic information

Entry
Database: PDB / ID: 9d5l
TitleThe C-terminal domain of Thiopseudomonas alkaliphila Tn7 TnsE bound to DNA
Components
  • DNA
  • TnsE, Tn7 transposition protein
KeywordsDNA BINDING PROTEIN/DNA / Tn7 / target-site selector / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homologyPHOSPHATE ION / DNA / DNA (> 10)
Function and homology information
Biological speciesThiopseudomonas alkaliphila (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsKrishnan, S.S. / Guarne, A.
Funding support Canada, 2items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-155941 Canada
Canadian Institutes of Health Research (CIHR)PJT-189946 Canada
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Asymmetric loading of TnsE regulates Tn7 targeting of DNA replication structures.
Authors: Shreya S Krishnan / Yao Shen / Treasa B O'Hagan / Lindsay A Matthews / Nuwani W Weerasinghe / Rodolfo Ghirlando / Christopher J Thibodeaux / Alba Guarné /
Abstract: Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a ...Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a conserved site in the chromosome or replicating DNA structures in conjugal plasmids, ensuring the vertical and horizontal spread of the element. While the pathway targeting the attTn7 site in the bacterial chromosome has been extensively studied, the pathway targeting DNA replication structures remains poorly understood. We have used an integrative structural biology approach to elucidate how the Tn7-encoded protein TnsE recognizes replication sites. Using native mass spectrometry, we found that TnsE forms 1:1 and 2:1 (TnsE:DNA) complexes on 3'-recessed DNA, with gain-of-function TnsE variants favoring the formation of 2:1 complexes. Structural characterization confirms that two TnsE molecules bind to DNA with the C-terminal domain of the protein recognizing duplex DNA, leaving the N-terminal domain to impose DNA substrate specificity and recruit the core transposition machinery. Collectively, our work is consistent with a model where TnsE-mediated target-site selection relies on the formation of an asymmetric TnsE:DNA complex to recruit the Tn7 transposase to DNA replication structures.
History
DepositionAug 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TnsE, Tn7 transposition protein
C: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,9853
Polymers22,8902
Non-polymers951
Water34219
1
A: TnsE, Tn7 transposition protein
C: DNA
hetero molecules

A: TnsE, Tn7 transposition protein
C: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,9716
Polymers45,7814
Non-polymers1902
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation12_555x,x-y,-z+1/61
Buried area4960 Å2
ΔGint-45 kcal/mol
Surface area18390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)63.770, 63.770, 220.510
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
Space group name HallP612(x,y,z+5/12)
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/6
#3: y,-x+y,z+5/6
#4: -y,x-y,z+1/3
#5: -x+y,-x,z+2/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+2/3
#8: -x,-y,z+1/2
#9: y,x,-z+1/3
#10: -y,-x,-z+5/6
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/6

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Components

#1: Protein TnsE, Tn7 transposition protein


Mass: 19230.975 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thiopseudomonas alkaliphila (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA


Mass: 3659.440 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The DNA used in the crystallization condition was a 3'-recessed DNA substrate formed by annealing a 48-nt (5'-GCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGG-3') and a 24-nt (5'- ...Details: The DNA used in the crystallization condition was a 3'-recessed DNA substrate formed by annealing a 48-nt (5'-GCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGG-3') and a 24-nt (5'-CCTAGTTTAGTTGGCTGATCGTTG-3') resulting in a 24 bp double-strand and 24 nt single-strand portion. The asymmetric unit contains one molecule of TaTnsE-CTD and a DNA strand (12 nt) that forms a DNA duplex by crystal symmetry and propagates as a pseudo-continuous duplex. This arrangement resulted in the asymmetric unit representing an average of the protein binding along the entire duplex, precluding the assignment of the DNA register and washing out the signal for the 5' single-strand DNA tail. We have modelled a string of T/A basepairs to reflect the lack of registry.
Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 19 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN
Sequence detailsThe DNA used in the crystallization condition was a 3'-recessed DNA substrate formed by annealing a ...The DNA used in the crystallization condition was a 3'-recessed DNA substrate formed by annealing a 48-nt (5'-GCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGG-3') and a 24-nt (5'-CCTAGTTTAGTTGGCTGATCGTTG-3') resulting in a 24 bp double-strand and 24 nt single-strand portion. The asymmetric unit contains one molecule of TaTnsE-CTD and a DNA strand (12 nt) that forms a DNA duplex by crystal symmetry and propagates as a pseudo-continuous duplex. This arrangement resulted in the asymmetric unit representing an average of the protein binding along the entire duplex, precluding the assignment of the DNA register and washing out the signal for the 5' single-strand DNA tail. We have modelled a string of T/A basepairs to reflect the lack of registry.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.83 Å3/Da / Density % sol: 56.49 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / Details: Sodium Iodide, Bis-Tris propane, PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08B1-1 / Wavelength: 1.0332 Å
DetectorType: RAYONIX MX300HE / Detector: CCD / Date: Jun 29, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 2.8→49.38 Å / Num. obs: 7169 / % possible obs: 99.6 % / Redundancy: 38.4 % / Biso Wilson estimate: 61.71 Å2 / CC1/2: 0.995 / Net I/σ(I): 9.03
Reflection shellResolution: 2.8→2.9 Å / Num. unique obs: 702 / CC1/2: 0.42

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHENIX1.18.2_3874phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.8→49.38 Å / SU ML: 0.498 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.5255
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2779 714 10 %
Rwork0.2368 6428 -
obs0.2403 7142 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 67.48 Å2
Refinement stepCycle: LAST / Resolution: 2.8→49.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1277 246 5 19 1547
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00621581
X-RAY DIFFRACTIONf_angle_d0.55782180
X-RAY DIFFRACTIONf_chiral_restr0.0384237
X-RAY DIFFRACTIONf_plane_restr0.0037237
X-RAY DIFFRACTIONf_dihedral_angle_d18.6892610
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8-3.020.43061360.39351232X-RAY DIFFRACTION99.35
3.02-3.320.31111390.2611242X-RAY DIFFRACTION99.21
3.32-3.80.27031390.23751259X-RAY DIFFRACTION99.71
3.8-4.790.23531430.19271291X-RAY DIFFRACTION99.93
4.79-49.380.27651570.23071404X-RAY DIFFRACTION99.81

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