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- EMDB-46582: Cryo-electron microscopy structure of TnsE-A453V/D523N bound to 3... -

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Basic information

Entry
Database: EMDB / ID: EMD-46582
TitleCryo-electron microscopy structure of TnsE-A453V/D523N bound to 3'-recessed DNA
Map dataUnsharpened cryo-EM map from cryosparc non-uniform refinement of sample containing TnsE-A453V/D523N bound to 3'-recessed DNA
Sample
  • Complex: TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA
  • Protein or peptide: Transposon Tn7 transposition protein TnsE-A453V/D523N
  • DNA: DNA (5'-CCAGGAGCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGGACATCT-3')
  • DNA: DNA (5'-AGATGTCCTAGTTTAGTTGGCTGATCGTTG-3')
KeywordsTn7 / target-site selector / DNA BINDING PROTEIN-DNA complex
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsKrishnan SS / Guarne A
Funding support Canada, 2 items
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 14, 2024-
Header (metadata) releaseAug 27, 2025-
Map releaseAug 27, 2025-
UpdateAug 27, 2025-
Current statusAug 27, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46582.png

Downloads & links

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Map

FileDownload / File: emd_46582.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened cryo-EM map from cryosparc non-uniform refinement of sample containing TnsE-A453V/D523N bound to 3'-recessed DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 384 pix.
= 259.2 Å		0.68 Å/pix.
x 384 pix.
= 259.2 Å		0.68 Å/pix.
x 384 pix.
= 259.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.675 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0073
Minimum - Maximum-0.03521442 - 0.13337556
Average (Standard dev.)-0.000050892875 (±0.0035726002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map B

Fileemd_46582_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_46582_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA

EntireName: TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA
Components
  • Complex: TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA
  • Protein or peptide: Transposon Tn7 transposition protein TnsE-A453V/D523N
  • DNA: DNA (5'-CCAGGAGCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGGACATCT-3')
  • DNA: DNA (5'-AGATGTCCTAGTTTAGTTGGCTGATCGTTG-3')

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Supramolecule #1: TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA

SupramoleculeName: TnsE-A453V/D523N bound to 30ss+30ds 3'-recessed DNA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 152 KDa

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Macromolecule #1: Transposon Tn7 transposition protein TnsE-A453V/D523N

MacromoleculeName: Transposon Tn7 transposition protein TnsE-A453V/D523N / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVRLATFNDN VQVVHIGHLF RNSGHKEWRI FVWFNPMQER KWTRFTHLPL LSRAKVVNST TKQINKADR VIEFEASDLQ RAKIIDFPNL SSFASVRNKD GAQSSFIYEA ETPYSKTRYH I PQLELARS LFLINSYFCR SCLSSTALQQ EFDVQYEVER DHLEIRILPS ...String:
MVRLATFNDN VQVVHIGHLF RNSGHKEWRI FVWFNPMQER KWTRFTHLPL LSRAKVVNST TKQINKADR VIEFEASDLQ RAKIIDFPNL SSFASVRNKD GAQSSFIYEA ETPYSKTRYH I PQLELARS LFLINSYFCR SCLSSTALQQ EFDVQYEVER DHLEIRILPS SSFPKGALEQ SA VVQLLVW LFSDQDVMDS YESIFRHYQQ NREIKNGVES WCFSFDPPPM QGWKLHVKGR SSN EDKDYL VEEIVGLEIN AMLPSTTAIS HASFQEKEAG DGSTQHIAVS TESVVDDEHL QLDD EETAN IDTDTRVIEA EPTWISFSRP SRIEKSRRAR KSSQTILEKE EATTSENSNL VSTDE PHLG GVLAAADVGG KQDATNYNSI FANRFAAFDE LLSILKTKFA CRVLFEETLV LPKVGR SRL HLCKDGSPRV IKAVGVQRNG SEFVLLEVDV SDGVKMLSTK VLSGVDSETW RNDFEKI RR GVVKSSLNWP NSLFDQLYGQ DGHRGVNHPK GLGELQVSRE NMEGWAERVV REQFTHLE H HHHHH

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Macromolecule #2: DNA (5'-CCAGGAGCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGGACA...

MacromoleculeName: DNA (5'-CCAGGAGCAAGGCCGGAAACGTCACCAATGCAACGATCAGCCAACTAAACTAGGACATCT-3')
type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CCAGGAGCAA GGCCGGAAAC GTCACCAATG CAACGATCAG CCAACTAAAC TAGGACATCT

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Macromolecule #3: DNA (5'-AGATGTCCTAGTTTAGTTGGCTGATCGTTG-3')

MacromoleculeName: DNA (5'-AGATGTCCTAGTTTAGTTGGCTGATCGTTG-3') / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
AGATGTCCTA GTTTAGTTGG CTGATCGTTG

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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

Concentration1.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris.HClTris
0.15 MNaClsodium chloride
1.4 mMBMEbeta-mercaptoethanol
0.1 mMEDTAEDTA
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7224 / Average exposure time: 2.0 sec. / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: OTHER / Details: Stochastic gradient descent
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 110312
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Avg.num./class: 40000 / Software - Name: cryoSPARC

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