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- EMDB-15848: IstA transposase cleaved donor complex -

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Basic information

Entry
Database: EMDB / ID: EMD-15848
TitleIstA transposase cleaved donor complex
Map data
Sample
  • Complex: IstA transposase cleaved donor complex
    • Protein or peptide: Putative transposase for insertion sequence element IS5376
    • DNA: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
    • DNA: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
  • Ligand: MAGNESIUM ION
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
Putative transposase for insertion sequence element IS5376
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsSpinola-Amilibia M / de la Gandara A / Araujo-Bazan L / 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 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, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15848.png

Downloads & links

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Map

FileDownload / File: emd_15848.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.16822992 - 0.19696657
Average (Standard dev.)4.0691775e-05 (±0.0038705005)
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_15848_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_15848_additional_1.map
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Half map: #1

Fileemd_15848_half_map_1.map
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Half map: #2

Fileemd_15848_half_map_2.map
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Sample components

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Entire : IstA transposase cleaved donor complex

EntireName: IstA transposase cleaved donor complex
Components
  • Complex: IstA transposase cleaved donor complex
    • Protein or peptide: Putative transposase for insertion sequence element IS5376
    • DNA: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
    • DNA: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: IstA transposase cleaved donor complex

SupramoleculeName: IstA transposase cleaved donor complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Details: Complex of IstA transposase bound to two right IS5376 transposon ends
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 225 KDa

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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
Details: The residues annotated as cloning artefact are the following five flanking bases of the X67861 gene
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 47.687723 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 KKQTEMAATI SPVSVEVDTR PLSVYDAFLR GESS ENLYF Q

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Macromolecule #2: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)

MacromoleculeName: DNA (57-MER) / right IS21 transposon end (insertion sequence IS5376)
type: dna / ID: 2 / Details: GB X67861 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 18.384814 KDa
SequenceString: (DA)(DT)(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) ...String:
(DA)(DT)(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)

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Macromolecule #3: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)

MacromoleculeName: DNA (55-MER) / right IS21 transposon end (insertion sequence IS5376)
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 17.029939 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)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.119 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMHEPESHEPES
150.0 mMNaClSodium chlorideSodium chloride
5.0 mMMgCl2Magnesium chloride
1.0 mMDTT1,4-Dithiothreitol
0.005 %Tween-20Tween-20
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.36 sec. / Average electron dose: 59.7 e/Å2 / Details: Single shot per hole
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 11094653
Startup modelType of model: OTHER
Details: Obtained, from cryo-EM data, using the Stochastic Gradient Descent (SGD) algorithm implemented in Relion 3.1.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 6 / Avg.num./class: 180000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 337376
DetailsSuper-resolution counting mode
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 115.818
Output model

PDB-8b4h:
IstA transposase cleaved donor complex

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