+Open data
-Basic information
Entry | Database: PDB / ID: 8q4d | |||||||||
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Title | IstA-IstB(E167Q) Strand Transfer Complex | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / DNA transposition / DNA integration / transposon / transpososome / holo-transpososome / insertion sequence / IS21 / IstA / IstB / DDE transposase / DDE domain / AAA+ ATPase / DNA strand transfer complex / STC | |||||||||
Function / homology | Function and homology information transposition / DNA strand exchange activity / DNA integration / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Geobacillus stearothermophilus (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å | |||||||||
Authors | de la Gandara, A. / Spinola-Amilibia, M. / Araujo-Bazan, L. / Nunez-Ramirez, R. / Berger, J.M. / Arias-Palomo, E. | |||||||||
Funding support | Spain, 2items
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Citation | Journal: Nature / Year: 2024 Title: Molecular basis for transposase activation by a dedicated AAA+ ATPase. Authors: Álvaro de la Gándara / Mercedes Spínola-Amilibia / Lidia Araújo-Bazán / Rafael Núñez-Ramírez / James M Berger / Ernesto Arias-Palomo / Abstract: Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that ...Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8q4d.cif.gz | 2.4 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8q4d.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8q4d.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8q4d_validation.pdf.gz | 2.6 MB | Display | wwPDB validaton report |
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Full document | 8q4d_full_validation.pdf.gz | 2.7 MB | Display | |
Data in XML | 8q4d_validation.xml.gz | 184.9 KB | Display | |
Data in CIF | 8q4d_validation.cif.gz | 274 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/q4/8q4d ftp://data.pdbj.org/pub/pdb/validation_reports/q4/8q4d | HTTPS FTP |
-Related structure data
Related structure data | 18144MC 8q3wC C: citing same article (ref.) M: map data used to model this data |
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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
-Protein , 2 types, 24 molecules ABCDEFGHIJKLMNOPQRSTUVWX
#1: Protein | Mass: 44012.723 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source 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 (E. coli) / Variant (production host): C41 / References: UniProt: Q45618 #2: Protein | Mass: 28817.422 Da / Num. of mol.: 20 Source method: isolated from a genetically manipulated source Details: E167Q mutant Source: (gene. exp.) Geobacillus stearothermophilus (bacteria) Plasmid: pHMKS Details (production host): TEV cleavable MBP-His6 tag at the N-terminus Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q45619 |
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-DNA chain , 1 types, 2 molecules ad
#3: DNA chain | Mass: 36524.391 Da / Num. of mol.: 2 / Source method: obtained synthetically Details: complete sequence of the right terminal inverted repeat (TIR) covalently bound to a target DNA Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: 1801971458 |
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-DNA (58-MER) / ... , 2 types, 4 molecules becf
#4: DNA chain | Mass: 17767.412 Da / Num. of mol.: 2 / Source method: obtained synthetically Details: complementary sequence of the right terminal inverted repeat (TIR) Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: 1190175513 #5: DNA chain | Mass: 17836.412 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: complementary sequence of the target DNA / Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: 1801971458 |
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-Non-polymers , 3 types, 40 molecules
#6: Chemical | ChemComp-MG / #7: Chemical | #8: Chemical | ChemComp-ATP / |
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-Details
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 |
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Molecular weight | Value: 0.952 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | |||||||||||||||||||||||||||||||||||
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: 1000 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.1 sec. / Electron dose: 51.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
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: 4102640 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 272218 / Num. of class averages: 3 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 91.21 / Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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