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- PDB-8q4d: IstA-IstB(E167Q) Strand Transfer Complex -

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Basic information

Entry
Database: PDB / ID: 8q4d
TitleIstA-IstB(E167Q) Strand Transfer Complex
Components
  • (DNA (58-MER) / ...) x 2
  • DNA (118-MER) / TIR-transferred strand
  • Insertion sequence IS5376 putative ATP-binding protein
  • Putative transposase for insertion sequence element IS5376
KeywordsDNA 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
HTH domain, IS21 transposase-type / IS21 transposase-type HTH domain profile. / : / DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / Resolvase, HTH domain / Helix-turn-helix domain of resolvase / ClpA/B family / Integrase core domain ...HTH domain, IS21 transposase-type / IS21 transposase-type HTH domain profile. / : / DNA replication protein DnaC/insertion sequence putative ATP-binding protein / IstB-like ATP-binding protein / IstB-like ATP binding protein / Resolvase, HTH domain / Helix-turn-helix domain of resolvase / ClpA/B family / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / : / : / DNA / DNA (> 10) / DNA (> 100) / Putative transposase for insertion sequence element IS5376 / Insertion sequence IS5376 putative ATP-binding protein
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 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 6, 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: 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: 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: Insertion sequence IS5376 putative ATP-binding protein
L: Insertion sequence IS5376 putative ATP-binding protein
M: Insertion sequence IS5376 putative ATP-binding protein
N: Insertion sequence IS5376 putative ATP-binding protein
O: Insertion sequence IS5376 putative ATP-binding protein
P: Insertion sequence IS5376 putative ATP-binding protein
Q: Insertion sequence IS5376 putative ATP-binding protein
R: Insertion sequence IS5376 putative ATP-binding protein
S: Insertion sequence IS5376 putative ATP-binding protein
T: Insertion sequence IS5376 putative ATP-binding protein
U: Insertion sequence IS5376 putative ATP-binding protein
V: Insertion sequence IS5376 putative ATP-binding protein
W: Insertion sequence IS5376 putative ATP-binding protein
X: Insertion sequence IS5376 putative ATP-binding protein
a: DNA (118-MER) / TIR-transferred strand
b: DNA (58-MER) / TIR non-transferred strand
c: DNA (58-MER) / target-reverse complement
d: DNA (118-MER) / TIR-transferred strand
e: DNA (58-MER) / TIR non-transferred strand
f: DNA (58-MER) / target-reverse complement
hetero molecules


Theoretical massNumber of molelcules
Total (without water)907,12670
Polymers896,65630
Non-polymers10,47040
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

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Protein , 2 types, 24 molecules ABCDEFGHIJKLMNOPQRSTUVWX

#1: Protein
Putative transposase for insertion sequence element IS5376


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
Insertion sequence IS5376 putative ATP-binding 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 DNA (118-MER) / TIR-transferred strand


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 DNA (58-MER) / TIR non-transferred strand


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 DNA (58-MER) / target-reverse complement


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 / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#8: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

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
1IstA-IstB(E167Q) Strand Transfer ComplexCOMPLEXIstA transposase and IstB(167Q) regulatory AAA+ ATPase bound to a strand transfer DNA#1-#50MULTIPLE SOURCES
2IstA-IstB(E167Q) StrandCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#51RECOMBINANT
Molecular weightValue: 0.952 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 (E. coli)562C41(DE3) and BL21(DE3)2-Cc-T and pHMKS vectors (pET derived)
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 triphosphateATP1
60.015 %Nonidet P-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: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 51.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9.8model fitting
9PHENIX1.19model refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
Image processingDetails: Super-resolution counting mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4102640
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 272218 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 91.21 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00365160
ELECTRON MICROSCOPYf_angle_d0.56489956
ELECTRON MICROSCOPYf_dihedral_angle_d17.62225326
ELECTRON MICROSCOPYf_chiral_restr0.0439844
ELECTRON MICROSCOPYf_plane_restr0.0039648

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