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- EMDB-18136: ATP-bound IstB in complex to duplex DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-18136
TitleATP-bound IstB in complex to duplex DNA
Map dataSharpened Map
Sample
  • Complex: IstB transposition regulator AAA+ ATPase bound to target DNA
    • Complex: Insertion sequence IS5376 putative ATP-binding protein
      • Protein or peptide: Insertion sequence IS5376 putative ATP-binding protein
    • Complex: DNA (48-MER) Target DNA FW and Rv
      • DNA: DNA (48-MER) Target DNA FW
      • DNA: DNA (48-MER) Traget DNA Rv
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsDNA Transposition / transposon / transpososome / AAA+ ATPases / target DNA / IS21 / IstB / Insertion sequence / DNA BINDING PROTEIN / 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
Insertion sequence IS5376 putative ATP-binding protein
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 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 4, 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_18136.png

Downloads & links

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Map

FileDownload / File: emd_18136.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.21831192 - 0.2675998
Average (Standard dev.)-0.000008997276 (±0.0050750994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 305.27997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18136_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened Map

Fileemd_18136_additional_1.map
AnnotationUnsharpened Map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Map used for modelling of amino-terminal domains after...

Fileemd_18136_additional_2.map
AnnotationMap used for modelling of amino-terminal domains after focused 3D classification
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18136_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18136_half_map_2.map
Projections & Slices
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Sample components

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Entire : IstB transposition regulator AAA+ ATPase bound to target DNA

EntireName: IstB transposition regulator AAA+ ATPase bound to target DNA
Components
  • Complex: IstB transposition regulator AAA+ ATPase bound to target DNA
    • Complex: Insertion sequence IS5376 putative ATP-binding protein
      • Protein or peptide: Insertion sequence IS5376 putative ATP-binding protein
    • Complex: DNA (48-MER) Target DNA FW and Rv
      • DNA: DNA (48-MER) Target DNA FW
      • DNA: DNA (48-MER) Traget DNA Rv
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: IstB transposition regulator AAA+ ATPase bound to target DNA

SupramoleculeName: IstB transposition regulator AAA+ ATPase bound to target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Complex of IstB transposition regulator AAA+ ATPase bound to target DNA
Molecular weightTheoretical: 337 KDa

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

SupramoleculeName: Insertion sequence IS5376 putative ATP-binding protein
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)

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Supramolecule #3: DNA (48-MER) Target DNA FW and Rv

SupramoleculeName: DNA (48-MER) Target DNA FW and Rv / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Geobacillus stearothermophilus (bacteria) / Synthetically produced: Yes

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

MacromoleculeName: Insertion sequence IS5376 putative ATP-binding protein
type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 29.602275 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPNMKERIHE YCHRLHLPVM AERWSAMAEY ASTHNISYSE FLFRLLEAEI VEKQARSIQT LIKLSKLPYR KTIDTFDFTA QPSVDERRI RELLTLSFID RKENILFLGP PGIGKTHLAI SIGMEAIARG YKTYFITAHD LVNQLRRADQ EGKLEKKLRV F VKPTVLII ...String:
GPNMKERIHE YCHRLHLPVM AERWSAMAEY ASTHNISYSE FLFRLLEAEI VEKQARSIQT LIKLSKLPYR KTIDTFDFTA QPSVDERRI RELLTLSFID RKENILFLGP PGIGKTHLAI SIGMEAIARG YKTYFITAHD LVNQLRRADQ EGKLEKKLRV F VKPTVLII DEMGYLKLDP NSAHYLFQVI ARRYEHAPII LTSNKSFGEW GEIVGDSVLA TAMLDRLLHH SIIFNLKGES YR LREKRLQ EEKQKDQ

UniProtKB: Insertion sequence IS5376 putative ATP-binding protein

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Macromolecule #2: DNA (48-MER) Target DNA FW

MacromoleculeName: DNA (48-MER) Target DNA FW / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 18.401746 KDa
SequenceString: (DT)(DG)(DC)(DT)(DT)(DG)(DC)(DG)(DA)(DT) (DG)(DA)(DT)(DC)(DC)(DG)(DA)(DC)(DG)(DT) (DG)(DT)(DT)(DA)(DG)(DC)(DC)(DA)(DC) (DG)(DC)(DT)(DG)(DA)(DC)(DT)(DA)(DG)(DT) (DT) (DA)(DT)(DG)(DC)(DC)(DA) ...String:
(DT)(DG)(DC)(DT)(DT)(DG)(DC)(DG)(DA)(DT) (DG)(DA)(DT)(DC)(DC)(DG)(DA)(DC)(DG)(DT) (DG)(DT)(DT)(DA)(DG)(DC)(DC)(DA)(DC) (DG)(DC)(DT)(DG)(DA)(DC)(DT)(DA)(DG)(DT) (DT) (DA)(DT)(DG)(DC)(DC)(DA)(DT)(DG) (DC)(DC)(DT)(DC)(DC)(DC)(DT)(DT)(DC)(DA) (DG)(DG)

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Macromolecule #3: DNA (48-MER) Traget DNA Rv

MacromoleculeName: DNA (48-MER) Traget DNA Rv / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 18.584918 KDa
SequenceString: (DC)(DC)(DT)(DG)(DA)(DA)(DG)(DG)(DG)(DA) (DG)(DG)(DC)(DA)(DT)(DG)(DG)(DC)(DA)(DT) (DA)(DA)(DC)(DT)(DA)(DG)(DT)(DC)(DA) (DG)(DC)(DG)(DT)(DG)(DG)(DC)(DT)(DA)(DA) (DC) (DA)(DC)(DG)(DT)(DC)(DG) ...String:
(DC)(DC)(DT)(DG)(DA)(DA)(DG)(DG)(DG)(DA) (DG)(DG)(DC)(DA)(DT)(DG)(DG)(DC)(DA)(DT) (DA)(DA)(DC)(DT)(DA)(DG)(DT)(DC)(DA) (DG)(DC)(DG)(DT)(DG)(DG)(DC)(DT)(DA)(DA) (DC) (DA)(DC)(DG)(DT)(DC)(DG)(DG)(DA) (DT)(DC)(DA)(DT)(DC)(DG)(DC)(DA)(DA)(DG) (DC)(DA)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 10 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 10 / 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 5-triphosphate
0.015 %NP-40NP-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: 5.0 sec. / Average electron dose: 60.0 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: 1.2 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: 2439865
Startup modelType of model: OTHER / Details: Obtained, from negative staining EM data
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 306745
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 6 / Avg.num./class: 230000 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 95.21
Output model

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

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