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

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Basic information

Entry
Database: PDB / ID: 7pik
TitleCryo-EM structure of E. coli TnsB in complex with right end fragment of Tn7 transposon
Components
  • (Right end fragment of Tn7 transposon) x 2
  • Transposon Tn7 transposition protein TnsB
KeywordsDNA BINDING PROTEIN / complex / nuclease / Tn7 / transposon
Function / homology
Function and homology information


transposase activity / DNA transposition / DNA integration / chromosome / DNA binding
Similarity search - Function
Ribonuclease H-like superfamily/Ribonuclease H / Integrase, catalytic core / Integrase catalytic domain profile. / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Transposon Tn7 transposition protein TnsB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsKaczmarska, Z. / Czarnocki-Cieciura, M. / Rawski, M. / Nowotny, M.
Funding support Poland, European Union, 2items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposon Tn7 transposition protein TnsB
B: Transposon Tn7 transposition protein TnsB
C: Transposon Tn7 transposition protein TnsB
D: Transposon Tn7 transposition protein TnsB
K: Right end fragment of Tn7 transposon
L: Right end fragment of Tn7 transposon
E: Transposon Tn7 transposition protein TnsB


Theoretical massNumber of molelcules
Total (without water)448,2837
Polymers448,2837
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26290 Å2
ΔGint-156 kcal/mol
Surface area100100 Å2

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Components

#1: Protein
Transposon Tn7 transposition protein TnsB


Mass: 81026.695 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: tnsB / Production host: Escherichia coli (E. coli) / References: UniProt: P13989
#2: DNA chain Right end fragment of Tn7 transposon


Mass: 21471.771 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain Right end fragment of Tn7 transposon


Mass: 21678.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1TnsB-DNA complexCOMPLEXTnsB from the canonical E. coli Tn7 element in complex with the right transposon end fragmentall0MULTIPLE SOURCES
2TnsBCOMPLEXTnsB from the canonical E. coli Tn7 element#11RECOMBINANT
3Tn7 transposon right end fragmentCOMPLEXE. coli Tn7 transposon right end fragment#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.286 MDaNO
210.081 MDaNO
310.043 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPESHEPES1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample fixed with 0.05% glutaraldehyde and concentrated prior to vitrification; exact concentration cannot be estimated accurately.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K / Details: 4 s blot time, -5 blot force.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPU2.1image acquisition
4cryoSPARC3.2CTF correction
7Coot0.9.4.1model fitting
12cryoSPARC3.23D reconstruction
13PHENIX1.18model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1141614 / Details: Filament Tracer in cryoSPARC
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 901496 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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