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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

Image control

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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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