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Yorodumi- PDB-7pik: Cryo-EM structure of E. coli TnsB in complex with right end fragm... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7pik | |||||||||
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Title | Cryo-EM structure of E. coli TnsB in complex with right end fragment of Tn7 transposon | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / complex / nuclease / Tn7 / transposon | |||||||||
Function / homology | Function and homology information transposase activity / DNA transposition / DNA integration / chromosome / DNA binding Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å | |||||||||
Authors | Kaczmarska, Z. / Czarnocki-Cieciura, M. / Rawski, M. / Nowotny, M. | |||||||||
Funding support | Poland, European Union, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7pik.cif.gz | 376.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7pik.ent.gz | 285.2 KB | Display | PDB format |
PDBx/mmJSON format | 7pik.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7pik_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7pik_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7pik_validation.xml.gz | 65.2 KB | Display | |
Data in CIF | 7pik_validation.cif.gz | 98.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pi/7pik ftp://data.pdbj.org/pub/pdb/validation_reports/pi/7pik | HTTPS FTP |
-Related structure data
Related structure data | 13439MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 | | Mass: 21471.771 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) #3: DNA chain | | Mass: 21678.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon | ||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K / Details: 4 s blot time, -5 blot force. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1141614 / Details: Filament Tracer in cryoSPARC | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 901496 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL |