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- EMDB-55638: Cryo-EM structure of the PseTnsAB paired-end complex (right end) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-55638
TitleCryo-EM structure of the PseTnsAB paired-end complex (right end) in the presence of Mg
Map data
Sample
  • Complex: PseTnsAB paired-end complex (right end) in the presence of Mg
    • Complex: PseTnsAB tetrameric complex with two Mg ions
      • Protein or peptide: PseTnsAB artificial fusion protein
    • Complex: Transposon DNA
      • DNA: Transposon right-end, transferred strand
      • DNA: Transposon right-end, non-transferred strand
  • Ligand: MAGNESIUM ION
KeywordsTransposase / DNA-binding / CRISPR-associated transposon / nuclease / DNA BINDING PROTEIN
Biological speciesPseudoalteromonas sp. S983 (bacteria) / Pseudoalteromonas (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsFinocchio G / Oberli S / 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: Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases.
Authors: Mateusz Walter / Giada Finocchio / Seraina Oberli / Iana C Hammerschmid / George D Lampe / Julia Karan / Thomas Swartjes / Samuel H Sternberg / Martin Jinek / Irma Querques
Abstract: CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for ...CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from e ( CAST) shows uniquely robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the CAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of TnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.
History
DepositionNov 11, 2025-
Header (metadata) releaseJun 3, 2026-
Map releaseJun 3, 2026-
UpdateJun 3, 2026-
Current statusJun 3, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55638.png

Downloads & links

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Map

FileDownload / File: emd_55638.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.1448039 - 0.37154436
Average (Standard dev.)-0.000059962476 (±0.007881368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_55638_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_55638_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : PseTnsAB paired-end complex (right end) in the presence of Mg

EntireName: PseTnsAB paired-end complex (right end) in the presence of Mg
Components
  • Complex: PseTnsAB paired-end complex (right end) in the presence of Mg
    • Complex: PseTnsAB tetrameric complex with two Mg ions
      • Protein or peptide: PseTnsAB artificial fusion protein
    • Complex: Transposon DNA
      • DNA: Transposon right-end, transferred strand
      • DNA: Transposon right-end, non-transferred strand
  • Ligand: MAGNESIUM ION

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Supramolecule #1: PseTnsAB paired-end complex (right end) in the presence of Mg

SupramoleculeName: PseTnsAB paired-end complex (right end) in the presence of Mg
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: PseTnsAB is an artificial fusion of PseTnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and PseTnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from Pseudoalteromonas sp. S983 ...Details: PseTnsAB is an artificial fusion of PseTnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and PseTnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from Pseudoalteromonas sp. S983 (NCBI Reference Sequence PNDL01000005.1) with a synthetic NLS linker (res 210-238)
Molecular weightTheoretical: 419 KDa

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Supramolecule #2: PseTnsAB tetrameric complex with two Mg ions

SupramoleculeName: PseTnsAB tetrameric complex with two Mg ions / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 390 KDa

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Supramolecule #3: Transposon DNA

SupramoleculeName: Transposon DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Pseudoalteromonas (bacteria)
Molecular weightTheoretical: 28 KDa

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Macromolecule #1: PseTnsAB artificial fusion protein

MacromoleculeName: PseTnsAB artificial fusion protein / type: protein_or_peptide / ID: 1
Details: Fusion of PseTnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and PseTnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from Pseudoalteromonas sp. S983 (NCBI Reference Sequence ...Details: Fusion of PseTnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and PseTnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from Pseudoalteromonas sp. S983 (NCBI Reference Sequence PNDL01000005.1) with a synthetic NLS linker (res 210-238)
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 97.719156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNMYIRNLRK PSPNKNVFKF ASTKVSSVVM CESSLEFDAC FHHEYNDLIE SFGSQPEGFK YEFMGKSLPY TPDALISYTD KTQKYHEYK PYSKIASPLF RAEFAAKRAA SLKLGIDLVL VTDRQIRVNP ILNNLKLLHR YSGVYGISGI QKELLSFIHK S GVIKLNDI ...String:
SNMYIRNLRK PSPNKNVFKF ASTKVSSVVM CESSLEFDAC FHHEYNDLIE SFGSQPEGFK YEFMGKSLPY TPDALISYTD KTQKYHEYK PYSKIASPLF RAEFAAKRAA SLKLGIDLVL VTDRQIRVNP ILNNLKLLHR YSGVYGISGI QKELLSFIHK S GVIKLNDI SSQVGIPIGE TRSFLFGLMH KGLVKADLGC DDLTNNPTLW ATPGSGSGKR TADGSEFESP KKKRKVGSGS GG MTDFFNE FDESLVPLKP QTPTQYVKLD DANLIQRDLD TFSDTFKNQA LQRYKLISTI DKKLSRGWTQ RNLDPILDEL FKG GDVVRP NWRTVARWRK KYIESNGDIA SLADKNHKMG NRTNRIKGDD KFFDKALERF LDAKRPTIAT AYQYYKDLIV IENE SIVEG KIPIISYNAF NKRIKAIPPY AVAVARHGKF KADQWFAYCA AHVPPTRILE RVEIDHTPLD LILLDDELLI PIGRP YLTL LIDVFSGCVL GFHLSYKSPS YVSAAKAITH AIKPKSLDAL NIELQNDWPC FGKFENLVVD NGAEFWSKNL EHACQS AGI NIQYNPVRKP WLKPFIERFF GVMNEYFLPE LPGKTFSNIL EKEEYKPEKD AIMRFSTFVE EFHRWIADVY HQDSNSR ET RIPIKRWQQG FDAYPPLTMN EEEETRFSML MRISDSRTLT RNGFKYQELM YDSTALADYR KHYPQTKETV KKLIKVDP D DISKIYVYLE ELESYLEVPC TDPTGYTDGL SIYEHKTIKK INREVIRESK DSLGLAKARM AIHERVKQEQ EVFIESKTK AKITAVKKQA QIADVSNTGT STIKVSEESA APVQKHISND NSDDWDDDLE AFE

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Macromolecule #2: Transposon right-end, transferred strand

MacromoleculeName: Transposon right-end, transferred strand / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 13.642771 KDa
SequenceString:
(DG)(DT)(DC)(DA)(DA)(DG)(DG)(DT)(DA)(DT) (DG)(DG)(DT)(DT)(DG)(DC)(DA)(DT)(DT)(DA) (DT)(DG)(DT)(DC)(DA)(DA)(DA)(DC)(DT) (DA)(DT)(DG)(DG)(DT)(DT)(DG)(DC)(DA)(DG) (DC) (DG)(DA)(DC)(DA)

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Macromolecule #3: Transposon right-end, non-transferred strand

MacromoleculeName: Transposon right-end, non-transferred strand / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Pseudoalteromonas sp. S983 (bacteria)
Molecular weightTheoretical: 14.711478 KDa
SequenceString:
(DA)(DT)(DA)(DG)(DT)(DG)(DT)(DC)(DG)(DC) (DT)(DG)(DC)(DA)(DA)(DC)(DC)(DA)(DT)(DA) (DG)(DT)(DT)(DT)(DG)(DA)(DC)(DA)(DT) (DA)(DA)(DT)(DG)(DC)(DA)(DA)(DC)(DC)(DA) (DT) (DA)(DC)(DC)(DT)(DT)(DG)(DA)(DC)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration2.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.24 mMC8H17KN2O4SHEPES potassium salt
8.06 mMC4H12ClNO3Tris hydrochloride
165.8 mMNaClsodium chloride
151.2 mMKClpotassium chloride
10.2 mMMgCl2magnesium chloride
0.6 mMC4H10O2S2dithiothreitol
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

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3440 / Average exposure time: 1.25 sec. / Average electron dose: 62.13 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: 1095968
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 model of monomeric PseTnsAB
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7)
Software - details: Non-Uniform Refinement was used fo final reconstruction
Number images used: 203418
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

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The initial model was derived from an AlphaFold3 prediction of the monomeric PseTnsAB fusion protein
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 93.2
Target criteria: Real-space map-model correlation and geometry restraints
Output model

PDB-9t7l:
Cryo-EM structure of the PseTnsAB paired-end complex (right end) in the presence of Mg

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