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- PDB-8b4h: IstA transposase cleaved donor complex -

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Basic information

Entry
Database: PDB / ID: 8b4h
TitleIstA transposase cleaved donor complex
Components
  • DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
  • DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
  • Putative transposase for insertion sequence element IS5376
KeywordsDNA 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
HTH domain, IS21 transposase-type / IS21 transposase-type HTH domain profile. / Resolvase, HTH domain / Helix-turn-helix domain of resolvase / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / Putative transposase for insertion sequence element IS5376
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsSpinola-Amilibia, M. / de la Gandara, A. / Araujo-Bazan, L. / Berger, J.M. / Arias-Palomo, E.
Funding support Spain, 2items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesPID2020-120275GB-I00 Spain
Spanish Ministry of Science, Innovation, and UniversitiesBFU2017-89143-P Spain
CitationJournal: 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
DepositionSep 20, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative transposase for insertion sequence element IS5376
B: Putative transposase for insertion sequence element IS5376
C: Putative transposase for insertion sequence element IS5376
D: Putative transposase for insertion sequence element IS5376
E: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
F: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
G: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
H: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,62910
Polymers261,5808
Non-polymers492
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38990 Å2
ΔGint-219 kcal/mol
Surface area104620 Å2
MethodPISA

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Components

#1: Protein
Putative transposase for insertion sequence element IS5376


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 DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)


Mass: 18384.814 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: GB X67861 / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#3: DNA chain DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)


Mass: 17029.939 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: X67861
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: 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
Molecular weightValue: 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 solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESHEPES1
2150 mMSodium chlorideNaClSodium chloride1
35 mMMagnesium chlorideMgCl21
41 mM1,4-DithiothreitolDTT1
50.005 %Tween-20Tween-201
SpecimenConc.: 0.119 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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 FIELDBright-field microscopy / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7Coot0.9.8model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19model refinement
Image processingDetails: Super-resolution counting mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11094653
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 337376 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 115.818 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00417406
ELECTRON MICROSCOPYf_angle_d0.69824374
ELECTRON MICROSCOPYf_dihedral_angle_d27.5783838
ELECTRON MICROSCOPYf_chiral_restr0.0382580
ELECTRON MICROSCOPYf_plane_restr0.0062308

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