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- PDB-30gb: Cryo-EM structure of the PseCascade-TniQ-TnsC complex bound to Ps... -

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Basic information

Entry
Database: PDB / ID: 30gb
TitleCryo-EM structure of the PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs
Components
  • AAA+ ATPase domain-containing protein
  • CRISPR RNA
  • Cas6
  • Cas7.1
  • Cas8
  • Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
  • Non-target DNA strand
  • Target DNA strand
  • TniQ.1
KeywordsDNA BINDING PROTEIN / Transposase / DNA-binding / RNA-binding / CRISPR-Cas / CRISPR-associated transposon
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex ...maintenance of CRISPR repeat elements / detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / outer membrane-bounded periplasmic space / endonuclease activity / periplasmic space / DNA damage response / membrane
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) / Maltose/Cyclodextrin ABC transporter, substrate-binding protein ...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) / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / : / Cas8 / Cas7.1 / Cas6 ...ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / : / Cas8 / Cas7.1 / Cas6 / TniQ.1 / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesPseudoalteromonas sp. S983 (bacteria)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 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 23, 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: Target DNA strand
3: Non-target DNA strand
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: AAA+ ATPase domain-containing protein
L: AAA+ ATPase domain-containing protein
M: AAA+ ATPase domain-containing protein
N: AAA+ ATPase domain-containing protein
O: AAA+ ATPase domain-containing protein
P: AAA+ ATPase domain-containing protein
Q: AAA+ ATPase domain-containing protein
R: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
S: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
T: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
U: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
V: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
W: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
X: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,818,84541
Polymers1,815,12427
Non-polymers3,72014
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Assembly of the complex by co-precipitation was verified by SDS-PAGE.
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 Target DNA strand


Mass: 37162.676 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Target DNA strand complementary to the crRNA spacer sequence.
Source: (synth.) Pseudoalteromonas sp. S983 (bacteria)
#3: DNA chain Non-target DNA strand


Mass: 36726.367 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 , 6 types, 24 molecules ABCDEFGHIJKLMNOPQRSTUVWX

#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
AAA+ ATPase domain-containing protein


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) / Gene: PARC_a0488 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A290S0Z4
#9: Protein
Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 142301.844 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Details: C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI ...Details: C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).,C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).,C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).
Source: (gene. exp.) Escherichia coli K-12 (bacteria), (gene. exp.) Pseudoalteromonas sp. S983 (bacteria)
Gene: malE, b4034, JW3994 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0AEX9

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Non-polymers , 2 types, 14 molecules

#10: 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
#11: 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: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs
Type: COMPLEX
Details: PseCascade-TniQ-TnsC complex bound to seven C-terminal PseTnsB-hooks. The CRISPR RNA (crRNA) is base-paired with a double-stranded DNA target. The hook comprises residues 605-613 of TnsB ...Details: PseCascade-TniQ-TnsC complex bound to seven C-terminal PseTnsB-hooks. The CRISPR RNA (crRNA) is base-paired with a double-stranded DNA target. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).
Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.74 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
3300 mMpotassium chlorideKCl1
41 mMdithiothreitolC4H10O2S21
50.025 %polysorbate 20 (Tween 20)C58H114O261
61 mMadenosin-5'-triphosphate disodium saltC10H14N5Na2O13P31
710 mMmaltoseC12H22O111
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was prepared by co-precipitation, using His-MBP-tagged TnsAB bound to a double-stranded right-end substrate as bait to capture Cascade-TniQ and TnsC.
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-PROPANE / 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: 60.95 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11374
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7particle selectionBlob Picker was used for particle selection
2EPU3.9image acquisition
4cryoSPARC4.7CTF correctionPatch CTF Estimation was used for CTF correction
7Coot0.9.4model fitting
9PHENIX2.0_5936model refinement
10cryoSPARC4.7initial Euler assignmentAb-Initio Reconstruction was used for initial angular assignment
11cryoSPARC4.7final Euler assignmentNon-Uniform Refinement was used fo final angular assignment
12cryoSPARC4.7classificationAb-Initio Reconstruction was used for final classification
13cryoSPARC4.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 329059
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138834 / Algorithm: FOURIER SPACE / 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: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00353983
ELECTRON MICROSCOPYf_angle_d0.53273713
ELECTRON MICROSCOPYf_dihedral_angle_d17.1868846
ELECTRON MICROSCOPYf_chiral_restr0.0398147
ELECTRON MICROSCOPYf_plane_restr0.0038783

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