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- EMDB-13440: Cryo-EM helical reconstruction of E. coli TnsB in complex with ri... -

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Basic information

Entry
Database: EMDB / ID: EMD-13440
TitleCryo-EM helical reconstruction of E. coli TnsB in complex with right end fragment of Tn7 transposon
Map dataSharpened and symmetrized
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 chain 1
      • DNA: Right end fragment of Tn7 transposon chain 2
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 3.79 Å
AuthorsCzarnocki-Cieciura M / Kaczmarska Z
Funding supportEuropean Union, 2 items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-20E7/16-00European Union
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-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13440.png

Downloads & links

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Map

FileDownload / File: emd_13440.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and symmetrized
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.8644319 - 1.3042682
Average (Standard dev.)0.0071694953 (±0.038564578)
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_13440_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_13440_additional_1.map
AnnotationRaw map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_13440_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_13440_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 : 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 chain 1
      • DNA: Right end fragment of Tn7 transposon chain 2

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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)
Recombinant expressionOrganism: 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-#3 / 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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMWQINEVVL FDNDPYRILA IEDGQVVWMQ ISADKGVPQA RAELLLMQYL DEGRLVRTDD P YVHLDLEE PSVDSVSFQK REEDYRKILP IINSKDRFDP KVRSELVEHV VQEHKVTKAT VY KLLRRYW QRGQTPNALI PDYKNSGAPG ERRSATGTAK IGRAREYGKG ...String:
SMWQINEVVL FDNDPYRILA IEDGQVVWMQ ISADKGVPQA RAELLLMQYL DEGRLVRTDD P YVHLDLEE PSVDSVSFQK REEDYRKILP IINSKDRFDP KVRSELVEHV VQEHKVTKAT VY KLLRRYW QRGQTPNALI PDYKNSGAPG ERRSATGTAK IGRAREYGKG EGTKVTPEIE RLF RLTIEK HLLNQKGTKT TVAYRRFVDL FAQYFPRIPQ EDYPTLRQFR YFYDREYPKA QRLK SRVKA GVYKKDVRPL SSTATSQALG PGSRYEIDAT IADIYLVDHH DRQKIIGRPT LYIVI DVFS RMITGFYIGF ENPSYVVAMQ AFVNACSDKT AICAQHDIEI SSSDWPCVGL PDVLLA DRG ELMSHQVEAL VSSFNVRVES APPRRGDAKG IVESTFRTLQ AEFKSFAPGI VEGSRIK SH GETDYRLDAS LSVFEFTQII LRTILFRNNH LVMDKYDRDA DFPTDLPSIP VQLWQWGM Q HRTGSLRAVE QEQLRVALLP RRKVSISSFG VNLWGLYYSG SEILREGWLQ RSTDIARPQ HLEAAYDPVL VDTIYLFPQV GSRVFWRCNL TERSRQFKGL SFWEVWDIQA QEKHNKANAK QDELTKRRE LEAFIQQTIQ KANKLTPSTT EPKSTRIKQI KTNKKEAVTS ERKKRAEHLK P SSSGDEAK VIPFNAVEAD DQEDYSLPTY VPELFQDPPE KDES

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Macromolecule #2: Right end fragment of Tn7 transposon chain 1

MacromoleculeName: Right end fragment of Tn7 transposon chain 1 / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
CTAGTTTAAG ACTTTATTGT CATAGTTTAG ATCTATTTTG TTCAGTTTAA GACTTTATTG TCCGCCCACA

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Macromolecule #3: Right end fragment of Tn7 transposon chain 2

MacromoleculeName: Right end fragment of Tn7 transposon chain 2 / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
TGTGGGCGGA CAATAAAGTC TTAAACTGAA CAAAATAGAT CTAAACTATG ACAATAAAGT CTTAAACTAG

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPESHEPES
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 1141614 / Software - Name: cryoSPARC (ver. 3.2) / Software - details: Filament Tracer (BETA) was used / Details: Filament Tracer in cryoSPARC
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2)
Startup modelType of model: NONE
Details: initial model was generated directly from particle stack with Helical Refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Helical parameters - Δz: 112.734 Å
Applied symmetry - Helical parameters - Δ&Phi: -167.484 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 450748
FSC plot (resolution estimation)

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