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- EMDB-57737: TnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC complex b... -

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Basic information

Entry
Database: EMDB / ID: EMD-57737
TitleTnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
Map data
Sample
  • Complex: PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
KeywordsTransposase / DNA-binding / RNA-binding / CRISPR-Cas / CRISPR-associated transposon / DNA BINDING PROTEIN
Biological speciesPseudoalteromonas sp. S983 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsFinocchio G / Oberli S / Schmitz M / Jinek M
Funding supportEuropean Union, Switzerland, 2 items
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, 2026-
Header (metadata) releaseJun 17, 2026-
Map releaseJun 17, 2026-
UpdateJun 17, 2026-
Current statusJun 17, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_57737.png

Downloads & links

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Map

FileDownload / File: emd_57737.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 600 pix.
= 390. Å		0.65 Å/pix.
x 600 pix.
= 390. Å		0.65 Å/pix.
x 600 pix.
= 390. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
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Contour LevelBy AUTHOR: 0.057
Minimum - Maximum-0.13313262 - 0.40735316
Average (Standard dev.)0.0008109587 (±0.009730451)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 390.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_57737_msk_1.map
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Half map: #2

Fileemd_57737_half_map_1.map
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Half map: #1

Fileemd_57737_half_map_2.map
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Sample components

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Entire : PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs

EntireName: PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
Components
  • Complex: PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs

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Supramolecule #1: PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs

SupramoleculeName: PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
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).
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 740 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H17KN2O4SHEPES potassium salt
10.0 mMMgCl2magnesium chloride
300.0 mMKClpotassium chloride
1.0 mMC4H10O2S2dithiothreitol
0.025 %C58H114O26polysorbate 20 (Tween 20)
1.0 mMC10H14N5Na2O13P3adenosin-5'-triphosphate disodium salt
10.0 mMC12H22O11maltose
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe 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.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 11374 / Average exposure time: 1.25 sec. / Average electron dose: 60.95 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 329059
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7)
Software - details: Patch CTF Estimation was used for CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold 3 models of the individual chains were rigid-body fitted into the cryo-EM density map.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7)
Software - details: Local Refinement was used fo final reconstruction
Number images used: 138834
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7)
Software - details: Ab-Initio Reconstruction was used for initial angular assignment
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7)
Software - details: Non-Uniform Refinement was used fo final angular assignment
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 4.7)
Software - details: Ab-Initio Reconstruction was used for final classification
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 83.7
Target criteria: Real-space map-model correlation and geometry restraints

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