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- PDB-9vtp: Target DNA-bound type I-F3 TniQ-Cascade of Vibrio parahaemolyticu... -

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Basic information

Entry
Database: PDB / ID: 9vtp
TitleTarget DNA-bound type I-F3 TniQ-Cascade of Vibrio parahaemolyticus in partial R-loop state
Components
  • (CRISPR-associated ...) x 2
  • Non-target strand DNA from Vibrio alginolyticus strain K08M3 plasmid pL300
  • Target strand DNA from Vibrio alginolyticus K08M03 plasmid pL300
  • TniQ
  • Type I-F CRISPR-associated protein Csy3
  • crRNA from Vibrio parahaemolyticus
KeywordsDNA BINDING PROTEIN / CRISPR-associated transposon
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity
Similarity search - Function
TniQ / TniQ / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
: / : / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated protein, Csy4 family / Type I-F CRISPR-associated protein Csy3 / CRISPR-associated protein Csy2 / TniQ domain-containing protein
Similarity search - Component
Biological speciesVibrio parahaemolyticus RIMD 2210633 (bacteria)
Vibrio alginolyticus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.68 Å
AuthorsIshihara, K. / Numata, T.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H02916 Japan
Japan Society for the Promotion of Science (JSPS)24H00505 Japan
Japan Society for the Promotion of Science (JSPS)23KJ1734 Japan
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.
Authors: Kazuki Ishihara / Shunsuke Matsumoto / Christoph Gerle / Chai C Gopalasingam / Hideki Shigematsu / Tsuyoshi Shirai / Tomoyuki Numata /
Abstract: Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA- ...Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.
History
DepositionJul 11, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 11, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Feb 11, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: crRNA from Vibrio parahaemolyticus
2: Target strand DNA from Vibrio alginolyticus K08M03 plasmid pL300
3: Non-target strand DNA from Vibrio alginolyticus strain K08M3 plasmid pL300
A: Type I-F CRISPR-associated protein Csy3
B: Type I-F CRISPR-associated protein Csy3
C: Type I-F CRISPR-associated protein Csy3
D: Type I-F CRISPR-associated protein Csy3
E: Type I-F CRISPR-associated protein Csy3
F: Type I-F CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy2
H: CRISPR-associated protein, Csy4 family
I: TniQ
J: TniQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)491,15714
Polymers491,13213
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules 23

#2: DNA chain Target strand DNA from Vibrio alginolyticus K08M03 plasmid pL300


Mass: 23027.676 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio alginolyticus (bacteria) / References: GenBank: 1190167432
#3: DNA chain Non-target strand DNA from Vibrio alginolyticus strain K08M3 plasmid pL300


Mass: 23224.912 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vibrio alginolyticus (bacteria) / References: GenBank: 1190167432

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Protein , 2 types, 8 molecules ABCDEFIJ

#4: Protein
Type I-F CRISPR-associated protein Csy3


Mass: 39455.414 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Gene: VPA1389 / Plasmid: pMAL-c2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: Q87GC8
#7: Protein TniQ


Mass: 47357.113 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Gene: VPA1387 / Plasmid: pMAL-c2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: Q87GD0

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CRISPR-associated ... , 2 types, 2 molecules GH

#5: Protein CRISPR-associated protein Csy2


Mass: 71108.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Chain g corresponds to an unassignable region of the Cas8/5 helical bundle in chain G (residues 326-389).
Source: (gene. exp.) Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Gene: VPA1388 / Plasmid: pMAL-c2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: Q87GC9
#6: Protein CRISPR-associated protein, Csy4 family


Mass: 22919.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Gene: VPA1390 / Plasmid: pMAL-c2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: Q87GC7

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RNA chain / Non-polymers , 2 types, 2 molecules 1

#1: RNA chain crRNA from Vibrio parahaemolyticus


Mass: 19405.658 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Plasmid: pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: GenBank: BA000032.2
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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: Vibrio parahaemolyticus type I-F3 TniQ-Cascade / Type: COMPLEX / Entity ID: #1-#4, #6-#7 / Source: RECOMBINANT
Molecular weightValue: 0.494 MDa / Experimental value: NO
Source (natural)Organism: Vibrio parahaemolyticus RIMD 2210633 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Gold CodonPlus RIL / Plasmid: pACYCDuet-1, pMAL-c2
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HCl1
2100 mMNaCl1
31 mMDTT1
45 mMMgCl21
50.001 %LMNG1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50.31 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of real images: 4906
EM imaging opticsEnergyfilter name: In-column Omega Filter

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Processing

EM software
IDName
1cryoSPARC
2SerialEM
3cryoSPARC
4UCSF
5CHIMERAX
6cryoSPARC
7cryoSPARC
8RELION
9cryoSPARC
10PHENIX
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.68 Å / Num. of particles: 31509 / Symmetry type: POINT
Atomic model building
ID 3D fitting-IDSource nameType
11SwissModelin silico model
21AlphaFoldin silico model
RefinementHighest resolution: 2.68 Å

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