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- EMDB-51458: CryoEM structure of PmcTnsC-dsDNA-AMPPNP in complex with PmcTnsAB hook -

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Basic information

Entry
Database: EMDB / ID: EMD-51458
TitleCryoEM structure of PmcTnsC-dsDNA-AMPPNP in complex with PmcTnsAB hook
Map data
Sample
  • Complex: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the TnsAB hook (TnsAB 892-898)
    • Protein or peptide: AAA+ ATPase domain-containing protein
    • Protein or peptide: Integrase
    • DNA: DNA
    • DNA: DNA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsAAA+ ATPase / Integrase / CRISPR / Tn7-like transposon / CAST / transposase / DNA BINDING PROTEIN
Function / homology
Function and homology information


DNA integration / nucleic acid binding / ATP hydrolysis activity
Similarity search - Function
TnsA endonuclease C-terminal / TnsA endonuclease, N-terminal / TnsA endonuclease C terminal / TnsA-like endonuclease N terminal / Transposase-like, Mu, C-terminal / Mu transposase, C-terminal / : / AAA domain / Integrase, catalytic core / Integrase catalytic domain profile. ...TnsA endonuclease C-terminal / TnsA endonuclease, N-terminal / TnsA endonuclease C terminal / TnsA-like endonuclease N terminal / Transposase-like, Mu, C-terminal / Mu transposase, C-terminal / : / AAA domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ORC1/DEAH AAA+ ATPase domain-containing protein / Integrase
Similarity search - Component
Biological speciesPeltigera membranacea (fungus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFinocchio G / Chanez C / Querques I / Speichert KJ / Jinek M
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons.
Authors: Giada Finocchio / Irma Querques / Christelle Chanez / Katarzyna J Speichert / Martin Jinek /
Abstract: CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated ...CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated action of the guide RNA-bound Cascade complex and the transposon proteins TniQ, TnsC, and TnsAB. The interaction between the transposase TnsAB and the ATPase TnsC is crucial for transposition activity, yet the underlying molecular details have remained elusive. Here, we investigate the type I-B CAST system from Peltigera membranacea cyanobiont. Cryo-electron microscopic structures of TnsC and its complex with the C-terminal region of TnsAB reveal that TnsC forms a heptameric ring that recruits TnsAB by interacting with its C-terminal tail. In vitro binding assays indicate that TnsAB exclusively interacts with the TnsC heptamer without inducing its disassembly, in contrast to type V-K CAST systems. Mutational analysis of key structural features corroborates the significance of TnsC multimerization and TnsB interaction for transposon activity in vivo. Altogether, these findings offer detailed structural and functional insights into the molecular mechanism of type I-B CAST, with the aim of facilitating their development as genome engineering tools.
History
DepositionAug 30, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51458.png

Downloads & links

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Map

FileDownload / File: emd_51458.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0361
Minimum - Maximum-0.10953415 - 0.2989248
Average (Standard dev.)0.00044369732 (±0.008691871)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_51458_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_51458_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the...

EntireName: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the TnsAB hook (TnsAB 892-898)
Components
  • Complex: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the TnsAB hook (TnsAB 892-898)
    • Protein or peptide: AAA+ ATPase domain-containing protein
    • Protein or peptide: Integrase
    • DNA: DNA
    • DNA: DNA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the...

SupramoleculeName: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the TnsAB hook (TnsAB 892-898)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Peltigera membranacea (fungus)

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Macromolecule #1: AAA+ ATPase domain-containing protein

MacromoleculeName: AAA+ ATPase domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Peltigera membranacea (fungus)
Molecular weightTheoretical: 43.794191 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMTKSTGF PLELLTRPAT ERLAYFENYT VAHPRLKEVY EILMRTIAEP AGASFIFVYG ASGVGKTTLR LRVEQKLTEL ALPKLESDR ARVPVVGIEA IAPESRYFNW KEYYTRALIT LEEPLIDHKF DYGVRGISRD NFGKINVESK VVAPALRRAL E NALIHRHP ...String:
SNAMTKSTGF PLELLTRPAT ERLAYFENYT VAHPRLKEVY EILMRTIAEP AGASFIFVYG ASGVGKTTLR LRVEQKLTEL ALPKLESDR ARVPVVGIEA IAPESRYFNW KEYYTRALIT LEEPLIDHKF DYGVRGISRD NFGKINVESK VVAPALRRAL E NALIHRHP DVFFVDEAQH FGKVASGYKL QDQLDCLKSL ANMTGILHCL LGTYELLTFR NLSGQLSRRS VDIHFRRYCA DS PEDVQAF KSVLLTFQQH LPLAETPNLV DHWEYFYERT LGCIGTLKDW LKRVLSDALD REATTITLKD LQKRALSVAQ CQK MFKEIQ EGERQLSETE ADVQNLRSAL GLGAKPIVLP EETPKTTRPP GKVGKRKPKR DPIGVQQDVS

UniProtKB: ORC1/DEAH AAA+ ATPase domain-containing protein

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Macromolecule #2: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Peltigera membranacea (fungus)
Molecular weightTheoretical: 5.207554 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SNAEGGKATT SRNEEPKLIQ VTFANTDFQA DEDEAIVPET LVVYEEF

UniProtKB: Integrase

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.898191 KDa
SequenceString:
(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT) (DG)(DC)(DA)(DT)(DG)(DC)

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Macromolecule #4: DNA

MacromoleculeName: DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.898191 KDa
SequenceString:
(DG)(DC)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DT)(DG)(DC)(DA)(DT)

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 7 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.48 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Model generated with AlphaFold2 ColabFold
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 24999
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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