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- EMDB-57728: TnsAB-focused cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB ho... -

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Basic information

Entry
Database: EMDB / ID: EMD-57728
TitleTnsAB-focused cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Map dataFocused map
Sample
  • Complex: PseCascade-TniQ-TnsC-TnsAB holocomplex
KeywordsTransposase / DNA-binding / RNA-binding / CRISPR-Cas / CRISPR-associated transposon / DNA BINDING PROTEIN
Biological speciesPseudoalteromonas sp. S983 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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_57728.png

Downloads & links

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Map

FileDownload / File: emd_57728.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 576 pix.
= 374.4 Å		0.65 Å/pix.
x 576 pix.
= 374.4 Å		0.65 Å/pix.
x 576 pix.
= 374.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033
Minimum - Maximum-0.103800476 - 0.22982562
Average (Standard dev.)0.0005953518 (±0.0061899666)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_57728_msk_1.map
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Additional map: Sharpened map

Fileemd_57728_additional_1.map
AnnotationSharpened map
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Half map: #1

Fileemd_57728_half_map_1.map
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Half map: #2

Fileemd_57728_half_map_2.map
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Sample components

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Entire : PseCascade-TniQ-TnsC-TnsAB holocomplex

EntireName: PseCascade-TniQ-TnsC-TnsAB holocomplex
Components
  • Complex: PseCascade-TniQ-TnsC-TnsAB holocomplex

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Supramolecule #1: PseCascade-TniQ-TnsC-TnsAB holocomplex

SupramoleculeName: PseCascade-TniQ-TnsC-TnsAB holocomplex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Details: PseCascade-TniQ-TnsC-TnsAB post-transposition holocomplex bound to a strand-transfer DNA substrate comprising a crRNA-matching target DNA fused to a double-stranded right-end DNA through a ...Details: PseCascade-TniQ-TnsC-TnsAB post-transposition holocomplex bound to a strand-transfer DNA substrate comprising a crRNA-matching target DNA fused to a double-stranded right-end DNA through a palindromic target site duplication.
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 1.2 MDa

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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

Concentration1.118 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 the DNA 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: 2 / Number real images: 43220 / Average exposure time: 1.25 sec. / Average electron dose: 62.82 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: 1919871
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 reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 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: 49606
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: Heterogeneous refinement was used for final classification

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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: 52.3
Target criteria: Real-space map-model correlation and geometry restraints

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