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- PDB-8q3w: ATP-bound IstB in complex to duplex DNA -

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Basic information

Entry
Database: PDB / ID: 8q3w
TitleATP-bound IstB in complex to duplex DNA
Components
  • DNA (48-MER) Target DNA FW
  • DNA (48-MER) Traget DNA Rv
  • Insertion sequence IS5376 putative ATP-binding protein
KeywordsDNA BINDING PROTEIN / DNA Transposition / transposon / transpososome / AAA+ ATPases / target DNA / IS21 / IstB / Insertion sequence / cryo-electron microscopy.
Function / homology
Function and homology information


DNA replication / ATP hydrolysis activity / ATP binding
Similarity search - Function
: / DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / ClpA/B family / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Insertion sequence IS5376 putative ATP-binding protein
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
Authorsde la Gandara, A. / Spinola-Amilibia, M. / Araujo-Bazan, L. / Nunez-Ramirez, R. / 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 UniversitiesPRE2018-086026 Spain
CitationJournal: 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
DepositionAug 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insertion sequence IS5376 putative ATP-binding protein
B: Insertion sequence IS5376 putative ATP-binding protein
C: Insertion sequence IS5376 putative ATP-binding protein
D: Insertion sequence IS5376 putative ATP-binding protein
E: Insertion sequence IS5376 putative ATP-binding protein
F: Insertion sequence IS5376 putative ATP-binding protein
G: Insertion sequence IS5376 putative ATP-binding protein
H: Insertion sequence IS5376 putative ATP-binding protein
I: Insertion sequence IS5376 putative ATP-binding protein
J: Insertion sequence IS5376 putative ATP-binding protein
K: DNA (48-MER) Target DNA FW
L: DNA (48-MER) Traget DNA Rv
hetero molecules


Theoretical massNumber of molelcules
Total (without water)338,32432
Polymers333,00912
Non-polymers5,31520
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Insertion sequence IS5376 putative ATP-binding protein


Mass: 29602.275 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
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
#2: DNA chain DNA (48-MER) Target DNA FW


Mass: 18401.746 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#3: DNA chain DNA (48-MER) Traget DNA Rv


Mass: 18584.918 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1IstB transposition regulator AAA+ ATPase bound to target DNACOMPLEXComplex of IstB transposition regulator AAA+ ATPase bound to target DNA#1-#30MULTIPLE SOURCES
2Insertion sequence IS5376 putative ATP-binding proteinCOMPLEX#11RECOMBINANT
3DNA (48-MER) Target DNA FW and RvCOMPLEX#2-#31SYNTHETIC
Molecular weightValue: 0.337 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Geobacillus stearothermophilus (bacteria)1422
33Geobacillus stearothermophilus (bacteria)1422
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)pHMKS
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMSodium chlorideNaCl1
35 mMMagnesium chlorideMgCl21
41 mM1,4-DithiothreitolDTT1
51 mMAdenosine 5-triphosphateATP1
60.015 %NP-40NP-401
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
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 FIELD / 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: 5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7Coot0.9.8model fitting
9PHENIX1.19model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
Image processingDetails: Super-resolution counting mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2439865
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 306745 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 95.21 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00223166
ELECTRON MICROSCOPYf_angle_d0.46331710
ELECTRON MICROSCOPYf_dihedral_angle_d13.2068864
ELECTRON MICROSCOPYf_chiral_restr0.0453514
ELECTRON MICROSCOPYf_plane_restr0.0033616

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