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- PDB-30gt: Cryo-EM structure of the PseCascade-TniQ-TnsC complex -

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Basic information

Entry
Database: PDB / ID: 30gt
TitleCryo-EM structure of the PseCascade-TniQ-TnsC complex
Components
  • CRISPR RNA
  • Cas6
  • Cas7.1
  • Cas8
  • NTS
  • TS
  • TniQ.1
  • TnsC
KeywordsDNA BINDING PROTEIN / Transposase / DNA-binding / RNA-binding / CRISPR-Cas / 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
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / Cas8 / Cas7.1 / Cas6 / TniQ.1
Similarity search - Component
Biological speciesPseudoalteromonas sp. S983 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFinocchio, G. / Oberli, S. / Schmitz, M. / Jinek, M.
Funding supportEuropean Union, Switzerland, 2items
OrganizationGrant numberCountry
European Research Council (ERC)820152European Union
Swiss National Science Foundation320030-228089 Switzerland
CitationJournal: bioRxiv / Year: 2026
Title: Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases.
Authors: Giada Finocchio / Seraina Oberli / George Lampe / Michael Schmitz / Samuel H Sternberg / Martin Jinek /
Abstract: CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like ...CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools. The type I-F CAST (CAST) displays the highest activity in mammalian cells to date and has been the subject of extensive directed evolution, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.
History
DepositionApr 24, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 17, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 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

Downloads & links

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Assembly

Deposited unit
1: CRISPR RNA
2: TS
3: NTS
A: Cas7.1
B: Cas7.1
C: Cas7.1
D: Cas7.1
E: Cas7.1
F: Cas7.1
G: Cas8
H: Cas6
I: TniQ.1
J: TniQ.1
K: TnsC
L: TnsC
M: TnsC
N: TnsC
O: TnsC
P: TnsC
Q: TnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)822,53434
Polymers818,81320
Non-polymers3,72014
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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RNA chain , 1 types, 1 molecules 1

#1: RNA chain CRISPR RNA


Mass: 30196.002 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CRISPR RNA (crRNA) from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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DNA chain , 2 types, 2 molecules 23

#2: DNA chain TS


Mass: 44605.438 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Target DNA strand complementary to the crRNA spacer sequence, fused to the transferred strand of the right transposon end via a palindromic target site duplication (TSD) sequence. TSD and ...Details: Target DNA strand complementary to the crRNA spacer sequence, fused to the transferred strand of the right transposon end via a palindromic target site duplication (TSD) sequence. TSD and right transposon end are unstructured.
Source: (synth.) Pseudoalteromonas sp. S983 (bacteria)
#3: DNA chain NTS


Mass: 29085.531 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Non-target DNA strand containing a +1 to +18 region identical to the target DNA strand, rather than complementary, to facilitate crRNA annealing to the protospacer.
Source: (synth.) Pseudoalteromonas sp. S983 (bacteria)

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Protein , 5 types, 17 molecules ABCDEFGHIJKLMNOPQ

#4: Protein
Cas7.1


Mass: 39845.145 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: CRISPR-associated protein Cas7 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983.
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0ABF7PQB9
#5: Protein Cas8


Mass: 79290.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CRISPR-associated protein Cas8 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983.
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0ABF7PQB8
#6: Protein Cas6


Mass: 26631.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CRISPR-associated protein Cas6 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983, bearing a C-terminal Twin-Strep tag.
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0ABF7PQC0
#7: Protein TniQ.1


Mass: 49832.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: CRISPR-associated protein TniQ from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, bearing an N-terminal His10 tag followed by a TEV protease cleavage site.
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0ABF7PQC1
#8: Protein
TnsC


Mass: 38609.902 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Details: AAA+ ATPase TnsC from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983.
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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Non-polymers , 2 types, 14 molecules

#9: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#10: 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 modificationY

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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: PseCascade-TniQ-TnsC complex / Type: COMPLEX
Details: PseCascade-TniQ-TnsC complex. The CRISPR RNA (crRNA) is base-paired with a double-stranded DNA target.
Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.73 MDa / Experimental value: NO
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES potassium saltC8H17KN2O4S1
210 mMmagnesium chlorideMgCl21
3150 mMpotassium chlorideKCl1
41 mMdithiothreitolC4H10O2S21
51 mMadenosin-5'-triphosphate disodium saltC10H14N5Na2O13P31
SpecimenConc.: 4.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was prepared by mixing and incubating the nucleic acid substrate and the protein components.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.25 sec. / Electron dose: 59.672 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9403
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.3particle selectionBlob Picker was used for particle selection
2EPU3.9image acquisition
4cryoSPARC4.3CTF correctionPatch CTF Estimation was used for CTF correction
7Coot0.9.4model fitting
9PHENIX2.0_5936model refinement
10cryoSPARC4.3initial Euler assignmentAb-Initio Reconstruction was used for initial angular assignment
11cryoSPARC4.3final Euler assignmentNon-Uniform Refinement was used fo final angular assignment
12cryoSPARC4.3classificationHeterogeneous refinement was used for final classification
13cryoSPARC4.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 745080
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76400 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Target criteria: Real-space map-model correlation and geometry restraints
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00453182
ELECTRON MICROSCOPYf_angle_d0.52672585
ELECTRON MICROSCOPYf_dihedral_angle_d16.9168742
ELECTRON MICROSCOPYf_chiral_restr0.0418038
ELECTRON MICROSCOPYf_plane_restr0.0038668

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