+Open data
-Basic information
Entry | Database: PDB / ID: 8b4h | |||||||||
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Title | IstA transposase cleaved donor complex | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / DNA Transposition / Transposase / Cleaved donor complex / DDE domain / IS21 / IstA / Insertion sequence | |||||||||
Function / homology | Function and homology information transposition / DNA strand exchange activity / DNA integration / DNA binding Similarity search - Function | |||||||||
Biological species | Geobacillus stearothermophilus (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å | |||||||||
Authors | Spinola-Amilibia, M. / de la Gandara, A. / Araujo-Bazan, L. / Berger, J.M. / Arias-Palomo, E. | |||||||||
Funding support | Spain, 2items
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Citation | Journal: Nat Commun / Year: 2023 Title: IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings. Authors: Mercedes Spínola-Amilibia / Lidia Araújo-Bazán / Álvaro de la Gándara / James M Berger / Ernesto Arias-Palomo / Abstract: Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use ...Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8b4h.cif.gz | 646.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8b4h.ent.gz | 531.5 KB | Display | PDB format |
PDBx/mmJSON format | 8b4h.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8b4h_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8b4h_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8b4h_validation.xml.gz | 57.9 KB | Display | |
Data in CIF | 8b4h_validation.cif.gz | 83.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/b4/8b4h ftp://data.pdbj.org/pub/pdb/validation_reports/b4/8b4h | HTTPS FTP |
-Related structure data
Related structure data | 15848MC 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: 47687.723 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Details: The residues annotated as cloning artefact are the following five flanking bases of the X67861 gene Source: (gene. exp.) Geobacillus stearothermophilus (bacteria) Plasmid: 2-Cc-T Details (production host): TEV cleavable MBP-His6 tag at the C-terminus Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: Q45618 #2: DNA chain | Mass: 18384.814 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: GB X67861 / Source: (synth.) Geobacillus stearothermophilus (bacteria) #3: DNA chain | Mass: 17029.939 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: X67861 #4: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: IstA transposase cleaved donor complex / Type: COMPLEX Details: Complex of IstA transposase bound to two right IS5376 transposon ends Entity ID: #1-#3 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.225 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Geobacillus stearothermophilus (bacteria) | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: C41(DE3) / Plasmid: 2-Cc-T vector (pET derived vector) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.119 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Details: 25 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K |
-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 defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3.36 sec. / Electron dose: 59.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Details: Single shot per hole |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Super-resolution counting mode | ||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 11094653 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 337376 / Num. of class averages: 2 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 115.818 / Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||
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