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- EMDB-18144: IstA-IstB(E167Q) Strand Transfer Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-18144
TitleIstA-IstB(E167Q) Strand Transfer Complex
Map datasharpened map
Sample
  • Complex: IstA-IstB(E167Q) Strand Transfer Complex
    • Complex: IstA-IstB(E167Q) Strand
      • Protein or peptide: Putative transposase for insertion sequence element IS5376
      • Protein or peptide: Insertion sequence IS5376 putative ATP-binding protein
    • Complex: DNA
      • DNA: DNA (118-MER) / TIR-transferred strand
      • DNA: DNA (58-MER) / TIR non-transferred strand
      • DNA: DNA (58-MER) / target-reverse complement
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsDNA transposition / DNA integration / transposon / transpososome / holo-transpososome / insertion sequence / IS21 / IstA / IstB / DDE transposase / DDE domain / AAA+ ATPase / DNA strand transfer complex / STC / DNA BINDING PROTEIN
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
Putative transposase for insertion sequence element IS5376 / Insertion sequence IS5376 putative ATP-binding protein
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.62 Å
Authorsde la Gandara A / Spinola-Amilibia M / Araujo-Bazan L / Nunez-Ramirez R / Berger JM / Arias-Palomo E
Funding support Spain, 2 items
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, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18144.png

Downloads & links

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Map

FileDownload / File: emd_18144.map.gz / Format: CCP4 / Size: 371.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 460 pix.
= 453.091 Å		0.98 Å/pix.
x 460 pix.
= 453.091 Å		0.98 Å/pix.
x 460 pix.
= 453.091 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.98498 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0037
Minimum - Maximum-0.020656021 - 0.05064255
Average (Standard dev.)0.00002263332 (±0.00089993683)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions460460460
Spacing460460460
CellA=B=C: 453.0908 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18144_msk_1.map
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Additional map: sharpened map of core-focused refinement, avoiding flexible regions

Fileemd_18144_additional_1.map
Annotationsharpened map of core-focused refinement, avoiding flexible regions
Projections & Slices
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Additional map: unsharpened map

Fileemd_18144_additional_2.map
Annotationunsharpened map
Projections & Slices
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Half map: #2

Fileemd_18144_half_map_1.map
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Half map: #1

Fileemd_18144_half_map_2.map
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Sample components

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Entire : IstA-IstB(E167Q) Strand Transfer Complex

EntireName: IstA-IstB(E167Q) Strand Transfer Complex
Components
  • Complex: IstA-IstB(E167Q) Strand Transfer Complex
    • Complex: IstA-IstB(E167Q) Strand
      • Protein or peptide: Putative transposase for insertion sequence element IS5376
      • Protein or peptide: Insertion sequence IS5376 putative ATP-binding protein
    • Complex: DNA
      • DNA: DNA (118-MER) / TIR-transferred strand
      • DNA: DNA (58-MER) / TIR non-transferred strand
      • DNA: DNA (58-MER) / target-reverse complement
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: IstA-IstB(E167Q) Strand Transfer Complex

SupramoleculeName: IstA-IstB(E167Q) Strand Transfer Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: IstA transposase and IstB(167Q) regulatory AAA+ ATPase bound to a strand transfer DNA
Molecular weightTheoretical: 952 KDa

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Supramolecule #2: IstA-IstB(E167Q) Strand

SupramoleculeName: IstA-IstB(E167Q) Strand / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#5
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)

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Macromolecule #1: Putative transposase for insertion sequence element IS5376

MacromoleculeName: Putative transposase for insertion sequence element IS5376
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 44.012723 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MITRGEFFMI KEMYERGMSI SDIARELGID RKTVRKYIHS PNPPSKSKRK QRKSKLDPFK PYLQKRMLED GVFNSEKLFF EIRQQGYTG GKTILKDYMK PFRETAKKKY TVRYETLPGE QMQVDWKEVG EVVIEGKKVK LSLFVATLGY SRMKYAVFTT S QDQEHLME ...String:
MITRGEFFMI KEMYERGMSI SDIARELGID RKTVRKYIHS PNPPSKSKRK QRKSKLDPFK PYLQKRMLED GVFNSEKLFF EIRQQGYTG GKTILKDYMK PFRETAKKKY TVRYETLPGE QMQVDWKEVG EVVIEGKKVK LSLFVATLGY SRMKYAVFTT S QDQEHLME CLIQSFKYFG GVPKKVLFDN MKTVTDGREQ GVVKWNQRFS EFASYYGFIP KVCRPYRAQT KGKVERAIQY IM DHFYVGT AFESIEELNF LLHRWLDQVA NRKPNATTGI SPQERWAEES LKPLPLKDYD TSYLSYRKVH WDGSFSYKGE QWL LSAEYA GKEILVKERL NGDIRLYFRG EEISHVDQQK KVISFAEKIK KKQTEMA

UniProtKB: Putative transposase for insertion sequence element IS5376

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Macromolecule #2: Insertion sequence IS5376 putative ATP-binding protein

MacromoleculeName: Insertion sequence IS5376 putative ATP-binding protein
type: protein_or_peptide / ID: 2 / Details: E167Q mutant / Number of copies: 20 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 28.817422 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: NMKERIHEYC HRLHLPVMAE RWSAMAEYAS THNISYSEFL FRLLEAEIVE KQARSIQTLI KLSKLPYRKT IDTFDFTAQP SVDERRIRE LLTLSFIDRK ENILFLGPPG IGKTHLAISI GMEAIARGYK TYFITAHDLV NQLRRADQEG KLEKKLRVFV K PTVLIIDQ ...String:
NMKERIHEYC HRLHLPVMAE RWSAMAEYAS THNISYSEFL FRLLEAEIVE KQARSIQTLI KLSKLPYRKT IDTFDFTAQP SVDERRIRE LLTLSFIDRK ENILFLGPPG IGKTHLAISI GMEAIARGYK TYFITAHDLV NQLRRADQEG KLEKKLRVFV K PTVLIIDQ MGYLKLDPNS AHYLFQVIAR RYEHAPIILT SNKSFGEWGE IVGDSVLATA MLDRLLHHSI IFNLKGESYR LR EKRLQEE

UniProtKB: Insertion sequence IS5376 putative ATP-binding protein

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Macromolecule #3: DNA (118-MER) / TIR-transferred strand

MacromoleculeName: DNA (118-MER) / TIR-transferred strand / type: dna / ID: 3
Details: complete sequence of the right terminal inverted repeat (TIR) covalently bound to a target DNA
Number of copies: 2 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 36.524391 KDa
SequenceString: (DC)(DC)(DT)(DT)(DC)(DT)(DG)(DG)(DG)(DG) (DA)(DA)(DT)(DT)(DT)(DT)(DA)(DA)(DA)(DC) (DC)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DT) (DT)(DG)(DG)(DG)(DG)(DA)(DA)(DA)(DA)(DA) (DA) (DT)(DA)(DA)(DT)(DC)(DG) ...String:
(DC)(DC)(DT)(DT)(DC)(DT)(DG)(DG)(DG)(DG) (DA)(DA)(DT)(DT)(DT)(DT)(DA)(DA)(DA)(DC) (DC)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DT) (DT)(DG)(DG)(DG)(DG)(DA)(DA)(DA)(DA)(DA) (DA) (DT)(DA)(DA)(DT)(DC)(DG)(DG)(DC) (DC)(DT)(DT)(DG)(DA)(DC)(DA)(DG)(DC)(DT) (DG)(DC) (DA)(DC)(DA)(DG)(DT)(DA)(DA) (DG)(DA)(DG)(DA)(DA)(DT)(DT)(DA)(DT)(DG) (DC)(DA)(DG) (DT)(DG)(DC)(DT)(DG)(DC) (DC)(DA)(DT)(DA)(DA)(DC)(DC)(DA)(DT)(DG) (DA)(DG)(DT)(DG) (DA)(DT)(DA)(DA)(DC) (DA)(DC)(DT)(DG)(DC)(DG)(DG)(DC)(DC)(DA) (DA)(DC)(DT)

GENBANK: GENBANK: MN043944.1

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Macromolecule #4: DNA (58-MER) / TIR non-transferred strand

MacromoleculeName: DNA (58-MER) / TIR non-transferred strand / type: dna / ID: 4
Details: complementary sequence of the right terminal inverted repeat (TIR)
Number of copies: 2 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 17.767412 KDa
SequenceString: (DT)(DC)(DA)(DT)(DG)(DT)(DC)(DA)(DA)(DG) (DG)(DC)(DC)(DG)(DA)(DT)(DT)(DA)(DT)(DT) (DT)(DT)(DT)(DT)(DC)(DC)(DC)(DC)(DA) (DA)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DG)(DG) (DT) (DT)(DT)(DA)(DA)(DA)(DA) ...String:
(DT)(DC)(DA)(DT)(DG)(DT)(DC)(DA)(DA)(DG) (DG)(DC)(DC)(DG)(DA)(DT)(DT)(DA)(DT)(DT) (DT)(DT)(DT)(DT)(DC)(DC)(DC)(DC)(DA) (DA)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DG)(DG) (DT) (DT)(DT)(DA)(DA)(DA)(DA)(DT)(DT) (DC)(DC)(DC)(DC)(DA)(DG)(DA)(DA)(DG)(DG)

GENBANK: GENBANK: CP020032.1

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Macromolecule #5: DNA (58-MER) / target-reverse complement

MacromoleculeName: DNA (58-MER) / target-reverse complement / type: dna / ID: 5 / Details: complementary sequence of the target DNA / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 17.836412 KDa
SequenceString: (DA)(DG)(DT)(DT)(DG)(DG)(DC)(DC)(DG)(DC) (DA)(DG)(DT)(DG)(DT)(DT)(DA)(DT)(DC)(DA) (DC)(DT)(DC)(DA)(DT)(DG)(DG)(DT)(DT) (DA)(DT)(DG)(DG)(DC)(DA)(DG)(DC)(DA)(DC) (DT) (DG)(DC)(DA)(DT)(DA)(DA) ...String:
(DA)(DG)(DT)(DT)(DG)(DG)(DC)(DC)(DG)(DC) (DA)(DG)(DT)(DG)(DT)(DT)(DA)(DT)(DC)(DA) (DC)(DT)(DC)(DA)(DT)(DG)(DG)(DT)(DT) (DA)(DT)(DG)(DG)(DC)(DA)(DG)(DC)(DA)(DC) (DT) (DG)(DC)(DA)(DT)(DA)(DA)(DT)(DT) (DC)(DT)(DC)(DT)(DT)(DA)(DC)(DT)(DG)(DT)

GENBANK: GENBANK: MN043944.1

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 20 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 18 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMNaClSodium chloride
5.0 mMMgCl2Magnesium chloride
1.0 mMDTT1,4-Dithiothreitol
1.0 mMATPAdenosine triphosphate
0.015 %NP-40Nonidet P-40
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 2.1 sec. / Average electron dose: 51.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsSuper-resolution counting mode
Particle selectionNumber selected: 4102640
Startup modelType of model: OTHER
Details: Obtained, from cryo-EM data, using the Stochastic Gradient Descent (SGD) algorithm implemented in Relion 4.0.
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 272218
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 68000 / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 91.21
Output model

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

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