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- PDB-30jw: Cryo-EM structure of the PseTnsAB paired-end complex (left end) i... -

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Basic information

Entry
Database: PDB / ID: 30jw
TitleCryo-EM structure of the PseTnsAB paired-end complex (left end) in the presence of Mg
Components
  • TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
  • Transposon left-end, non-transferred strand
  • Transposon left-end, transferred strand
KeywordsDNA BINDING PROTEIN / Transposase / DNA-binding / CRISPR-associated transposon / nuclease
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesPseudoalteromonas sp. S983 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsFinocchio, G. / Oberli, S. / 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: 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
DepositionApr 29, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 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

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Assembly

Deposited unit
A: TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
B: TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
C: TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
D: TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
E: Transposon left-end, transferred strand
F: Transposon left-end, non-transferred strand
G: Transposon left-end, transferred strand
H: Transposon left-end, non-transferred strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)447,65610
Polymers447,6078
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
TnsA endonuclease N-terminal domain-containing protein,TnsB transposase


Mass: 97719.156 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
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),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)
Source: (gene. exp.) Pseudoalteromonas sp. S983 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: DNA chain Transposon left-end, transferred strand


Mass: 13505.660 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Pseudoalteromonas sp. S983 (bacteria)
#3: DNA chain Transposon left-end, non-transferred strand


Mass: 14859.578 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Pseudoalteromonas sp. S983 (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeDetails (eV)Entity IDParent-IDSource
1PseTnsAB paired-end complex (left end) in the presence of MgCOMPLEXPseTnsAB 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)#1-#30MULTIPLE SOURCES
2PseTnsAB tetrameric complex with two Mg ionsCOMPLEX#11RECOMBINANT
3Transposon DNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.419 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudoalteromonas sp. S983 (bacteria)579572
33Pseudoalteromonas sp. S983 (bacteria)579572
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110.24 mMHEPES potassium saltC8H17KN2O4S1
28.06 mMTris hydrochlorideC4H12ClNO31
3165.8 mMsodium chlorideNaCl1
4151.2 mMpotassium chlorideKCl1
510.2 mMmagnesium chlorideMgCl21
60.6 mMdithiothreitolC4H10O2S21
SpecimenConc.: 6.04 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 2600 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.25 sec. / Electron dose: 64.201 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15223

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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
9PHENIX1.21.2_5419model 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
12cryoSPARC4.7classificationHeterogeneous Refinement was used for final classification
13cryoSPARC4.73D reconstructionNon-Uniform Refinement was used fo final reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4935000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 575675 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 93.9 / Protocol: FLEXIBLE FIT / Space: REAL
Target criteria: Real-space map-model correlation and geometry restraints
Atomic model buildingPDB-ID: 9T7L

9t7l


Accession code: 9T7L / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.51 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318630
ELECTRON MICROSCOPYf_angle_d0.59125738
ELECTRON MICROSCOPYf_dihedral_angle_d22.1593396
ELECTRON MICROSCOPYf_chiral_restr0.0392804
ELECTRON MICROSCOPYf_plane_restr0.0042862

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