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- EMDB-13439: Cryo-EM structure of E. coli TnsB in complex with right end fragm... -

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Basic information

Entry
Database: EMDB / ID: EMD-13439
TitleCryo-EM structure of E. coli TnsB in complex with right end fragment of Tn7 transposon
Map dataPrimary map used for model generation; sharpened with B-factor of -70 and after Local Filtering
Sample
  • Complex: TnsB-DNA complex
    • Complex: TnsB
      • Protein or peptide: Transposon Tn7 transposition protein TnsB
    • Complex: Tn7 transposon right end fragment
      • DNA: Right end fragment of Tn7 transposon
    • DNA: Right end fragment of Tn7 transposon
Keywordscomplex / nuclease / DNA binding protein / Tn7 / transposon
Function / homology
Function and homology information


transposase activity / DNA transposition / DNA integration / chromosome / DNA binding
Similarity search - Function
Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Transposon Tn7 transposition protein TnsB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsKaczmarska Z / Czarnocki-Cieciura M
Funding support Poland, European Union, 2 items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-20E7/16-00 Poland
European Union (EU)POIG.02.02.00-14-024/08-00European Union
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of transposon end recognition explains central features of Tn7 transposition systems.
Authors: Zuzanna Kaczmarska / Mariusz Czarnocki-Cieciura / Karolina M Górecka-Minakowska / Robert J Wingo / Justyna Jackiewicz / Weronika Zajko / Jarosław T Poznański / Michał Rawski / Timothy ...Authors: Zuzanna Kaczmarska / Mariusz Czarnocki-Cieciura / Karolina M Górecka-Minakowska / Robert J Wingo / Justyna Jackiewicz / Weronika Zajko / Jarosław T Poznański / Michał Rawski / Timothy Grant / Joseph E Peters / Marcin Nowotny /
Abstract: Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of ...Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.
History
DepositionAug 20, 2021-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13439.png

Downloads & links

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Map

FileDownload / File: emd_13439.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map used for model generation; sharpened with B-factor of -70 and after Local Filtering
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 512 pix.
= 440.32 Å		0.86 Å/pix.
x 512 pix.
= 440.32 Å		0.86 Å/pix.
x 512 pix.
= 440.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.9236138 - 5.4858685
Average (Standard dev.)0.0014117248 (±0.04463585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 440.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13439_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw map

Fileemd_13439_additional_1.map
AnnotationRaw map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_13439_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_13439_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TnsB-DNA complex

EntireName: TnsB-DNA complex
Components
  • Complex: TnsB-DNA complex
    • Complex: TnsB
      • Protein or peptide: Transposon Tn7 transposition protein TnsB
    • Complex: Tn7 transposon right end fragment
      • DNA: Right end fragment of Tn7 transposon
    • DNA: Right end fragment of Tn7 transposon

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Supramolecule #1: TnsB-DNA complex

SupramoleculeName: TnsB-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: TnsB from the canonical E. coli Tn7 element in complex with the right transposon end fragment
Molecular weightTheoretical: 43 KDa

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Supramolecule #2: TnsB

SupramoleculeName: TnsB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: TnsB from the canonical E. coli Tn7 element
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: Tn7 transposon right end fragment

SupramoleculeName: Tn7 transposon right end fragment / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: E. coli Tn7 transposon right end fragment
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Transposon Tn7 transposition protein TnsB

MacromoleculeName: Transposon Tn7 transposition protein TnsB / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 81.026695 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMWQINEVVL FDNDPYRILA IEDGQVVWMQ ISADKGVPQA RAELLLMQYL DEGRLVRTDD PYVHLDLEEP SVDSVSFQKR EEDYRKILP IINSKDRFDP KVRSELVEHV VQEHKVTKAT VYKLLRRYWQ RGQTPNALIP DYKNSGAPGE RRSATGTAKI G RAREYGKG ...String:
SMWQINEVVL FDNDPYRILA IEDGQVVWMQ ISADKGVPQA RAELLLMQYL DEGRLVRTDD PYVHLDLEEP SVDSVSFQKR EEDYRKILP IINSKDRFDP KVRSELVEHV VQEHKVTKAT VYKLLRRYWQ RGQTPNALIP DYKNSGAPGE RRSATGTAKI G RAREYGKG EGTKVTPEIE RLFRLTIEKH LLNQKGTKTT VAYRRFVDLF AQYFPRIPQE DYPTLRQFRY FYDREYPKAQ RL KSRVKAG VYKKDVRPLS STATSQALGP GSRYEIDATI ADIYLVDHHD RQKIIGRPTL YIVIDVFSRM ITGFYIGFEN PSY VVAMQA FVNACSDKTA ICAQHDIEIS SSDWPCVGLP DVLLADRGEL MSHQVEALVS SFNVRVESAP PRRGDAKGIV ESTF RTLQA EFKSFAPGIV EGSRIKSHGE TDYRLDASLS VFEFTQIILR TILFRNNHLV MDKYDRDADF PTDLPSIPVQ LWQWG MQHR TGSLRAVEQE QLRVALLPRR KVSISSFGVN LWGLYYSGSE ILREGWLQRS TDIARPQHLE AAYDPVLVDT IYLFPQ VGS RVFWRCNLTE RSRQFKGLSF WEVWDIQAQE KHNKANAKQD ELTKRRELEA FIQQTIQKAN KLTPSTTEPK STRIKQI KT NKKEAVTSER KKRAEHLKPS SSGDEAKVIP FNAVEADDQE DYSLPTYVPE LFQDPPEKDE S

UniProtKB: Transposon Tn7 transposition protein TnsB

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Macromolecule #2: Right end fragment of Tn7 transposon

MacromoleculeName: Right end fragment of Tn7 transposon / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21.471771 KDa
SequenceString: (DC)(DT)(DA)(DG)(DT)(DT)(DT)(DA)(DA)(DG) (DA)(DC)(DT)(DT)(DT)(DA)(DT)(DT)(DG)(DT) (DC)(DA)(DT)(DA)(DG)(DT)(DT)(DT)(DA) (DG)(DA)(DT)(DC)(DT)(DA)(DT)(DT)(DT)(DT) (DG) (DT)(DT)(DC)(DA)(DG)(DT) ...String:
(DC)(DT)(DA)(DG)(DT)(DT)(DT)(DA)(DA)(DG) (DA)(DC)(DT)(DT)(DT)(DA)(DT)(DT)(DG)(DT) (DC)(DA)(DT)(DA)(DG)(DT)(DT)(DT)(DA) (DG)(DA)(DT)(DC)(DT)(DA)(DT)(DT)(DT)(DT) (DG) (DT)(DT)(DC)(DA)(DG)(DT)(DT)(DT) (DA)(DA)(DG)(DA)(DC)(DT)(DT)(DT)(DA)(DT) (DT)(DG) (DT)(DC)(DC)(DG)(DC)(DC)(DC) (DA)(DC)(DA)

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Macromolecule #3: Right end fragment of Tn7 transposon

MacromoleculeName: Right end fragment of Tn7 transposon / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21.678018 KDa
SequenceString: (DT)(DG)(DT)(DG)(DG)(DG)(DC)(DG)(DG)(DA) (DC)(DA)(DA)(DT)(DA)(DA)(DA)(DG)(DT)(DC) (DT)(DT)(DA)(DA)(DA)(DC)(DT)(DG)(DA) (DA)(DC)(DA)(DA)(DA)(DA)(DT)(DA)(DG)(DA) (DT) (DC)(DT)(DA)(DA)(DA)(DC) ...String:
(DT)(DG)(DT)(DG)(DG)(DG)(DC)(DG)(DG)(DA) (DC)(DA)(DA)(DT)(DA)(DA)(DA)(DG)(DT)(DC) (DT)(DT)(DA)(DA)(DA)(DC)(DT)(DG)(DA) (DA)(DC)(DA)(DA)(DA)(DA)(DT)(DA)(DG)(DA) (DT) (DC)(DT)(DA)(DA)(DA)(DC)(DT)(DA) (DT)(DG)(DA)(DC)(DA)(DA)(DT)(DA)(DA)(DA) (DG)(DT) (DC)(DT)(DT)(DA)(DA)(DA)(DC) (DT)(DA)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPESHEPES
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4 s blot time, -5 blot force..
DetailsSample fixed with 0.05% glutaraldehyde and concentrated prior to vitrification; exact concentration cannot be estimated accurately.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 1141614 / Details: Filament Tracer in cryoSPARC
Startup modelType of model: NONE
Details: initial model was generated directly from particle stack with Helical Refinement
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 901496
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7pik:
Cryo-EM structure of E. coli TnsB in complex with right end fragment of Tn7 transposon

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