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- PDB-9gmz: CryoEM structure of PmcTnsC-dsDNA-AMPPNP in complex with PmcTnsAB hook -

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Basic information

Entry
Database: PDB / ID: 9gmz
TitleCryoEM structure of PmcTnsC-dsDNA-AMPPNP in complex with PmcTnsAB hook
Components
  • (DNA) x 2
  • AAA+ ATPase domain-containing protein
  • Integrase
KeywordsDNA BINDING PROTEIN / AAA+ ATPase / Integrase / CRISPR / Tn7-like transposon / CAST / transposase
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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / ORC1/DEAH AAA+ ATPase domain-containing protein / Integrase
Similarity search - Component
Biological speciesPeltigera membranacea (fungus)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFinocchio, G. / Chanez, C. / Querques, I. / Speichert, K.J. / Jinek, M.
Funding supportEuropean Union, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 9, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AAA+ ATPase domain-containing protein
B: AAA+ ATPase domain-containing protein
C: AAA+ ATPase domain-containing protein
D: AAA+ ATPase domain-containing protein
E: AAA+ ATPase domain-containing protein
F: AAA+ ATPase domain-containing protein
G: AAA+ ATPase domain-containing protein
H: Integrase
I: Integrase
J: Integrase
K: Integrase
L: Integrase
M: Integrase
1: DNA
2: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,31529
Polymers347,60115
Non-polymers3,71414
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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DNA chain , 2 types, 2 molecules 12

#3: DNA chain DNA


Mass: 4898.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain DNA


Mass: 4898.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / Protein/peptide , 2 types, 13 molecules ABCDEFGHIJKLM

#1: Protein
AAA+ ATPase domain-containing protein


Mass: 43794.191 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Gene: CDG76_08990 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFM2
#2: Protein/peptide
Integrase


Mass: 5207.554 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Peltigera membranacea (fungus) / Gene: CDG76_08995 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IFR8

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Non-polymers , 2 types, 14 molecules

#5: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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

ComponentName: TnsC heptameric ring bound to a dsDNA, AMPPNP and 6 copies of the TnsAB hook (TnsAB 892-898)
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Peltigera membranacea (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60.48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24999 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00220962
ELECTRON MICROSCOPYf_angle_d0.50728558
ELECTRON MICROSCOPYf_dihedral_angle_d12.9953280
ELECTRON MICROSCOPYf_chiral_restr0.0383197
ELECTRON MICROSCOPYf_plane_restr0.0033530

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