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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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