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- PDB-9t56: Cryo-EM structure of ISCro4-DBL-TBL-tDNA-dDNA synaptic complex -

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Basic information

Entry
Database: PDB / ID: 9t56
TitleCryo-EM structure of ISCro4-DBL-TBL-tDNA-dDNA synaptic complex
Components
  • (donor DNA (dDNA) ...) x 2
  • Donor-binding loop (DBL) RNA
  • ISCro4 transposase
  • Target-binding loop (TBL) RNA
  • target DNA (tDNA) bottom strand
  • target DNA (tRNA) top strand
KeywordsRECOMBINATION / Nucleic acid interaction / RNA binding / DNA binding / tetramer / recombinase
Function / homology
Function and homology information


transposase activity / DNA transposition / DNA binding
Similarity search - Function
Transposase, IS111A/IS1328/IS1533, N-terminal / : / Transposase / Transposase, IS116/IS110/IS902 / Transposase IS116/IS110/IS902 family
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / ISCro4 transposase
Similarity search - Component
Biological speciesCitrobacter rodentium ICC168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsFernandez Carrera, J. / Pelea, O. / Gerecke, S.E. / Chanez, C. / Jinek, M.
Funding supportEuropean Union, Switzerland, 2items
OrganizationGrant numberCountry
European Research Council (ERC)820152European Union
Swiss National Science Foundation320030-228089 Switzerland
CitationJournal: Science / Year: 2026
Title: Programmable genome editing in human cells using RNA-guided bridge recombinases.
Authors: Oana Pelea / András Tálas / Javier Fernández Carrera / Nicolas Mathis / Lilly van de Venn / Charles D Yeh / Péter I Kulcsár / Kim F Marquart / Yanik Weber / Saskia E Gerecke / Isabelle ...Authors: Oana Pelea / András Tálas / Javier Fernández Carrera / Nicolas Mathis / Lilly van de Venn / Charles D Yeh / Péter I Kulcsár / Kim F Marquart / Yanik Weber / Saskia E Gerecke / Isabelle F Harvey-Seutcheu / Dominic Mailänder / Moritz M Pfleiderer / Christelle Chanez / Jacob E Corn / Gerald Schwank / Martin Jinek /
Abstract: Site-specific insertion of gene-sized DNA fragments remains an unmet need in the genome editing field. IS110-family serine recombinases have recently been shown to mediate programmable DNA ...Site-specific insertion of gene-sized DNA fragments remains an unmet need in the genome editing field. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. Here, we show that the bridge recombinase ISCro4 is highly active in human cells, and provide structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multi-kilobase exisions and inversions, and facilitates donor DNA insertion at genomic sites with efficiencies exceeding 6%. Finally, we assess ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.
History
DepositionNov 4, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 18, 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

Downloads & links

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Assembly

Deposited unit
A: ISCro4 transposase
B: ISCro4 transposase
C: ISCro4 transposase
D: ISCro4 transposase
E: Target-binding loop (TBL) RNA
F: Donor-binding loop (DBL) RNA
G: target DNA (tRNA) top strand
H: target DNA (tDNA) bottom strand
I: donor DNA (dDNA) top strand
J: donor DNA (dDNA) bottom strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,02412
Polymers263,97510
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

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RNA chain , 2 types, 2 molecules EF

#2: RNA chain Target-binding loop (TBL) RNA


Mass: 35682.082 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: GGG added the 5'-end and UUUU appended at the 3'-end to facilitate in vitro transcription
Source: (synth.) Citrobacter rodentium ICC168 (bacteria) / References: GenBank: 282947233
#3: RNA chain Donor-binding loop (DBL) RNA


Mass: 25278.945 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: GGG added the 5'-end and UUUUUU appended at the 3'-end to facilitate in vitro transcription
Source: (synth.) Citrobacter rodentium ICC168 (bacteria)

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DNA chain , 2 types, 2 molecules GH

#4: DNA chain target DNA (tRNA) top strand


Mass: 11616.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Citrobacter rodentium ICC168 (bacteria)
#5: DNA chain target DNA (tDNA) bottom strand


Mass: 11776.551 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Citrobacter rodentium ICC168 (bacteria)

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Donor DNA (dDNA) ... , 2 types, 2 molecules IJ

#6: DNA chain donor DNA (dDNA) top strand


Mass: 13562.724 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Citrobacter rodentium ICC168 (bacteria)
#7: DNA chain donor DNA (dDNA) bottom strand


Mass: 13531.716 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Citrobacter rodentium ICC168 (bacteria)

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Protein / Non-polymers , 2 types, 6 molecules ABCD

#1: Protein
ISCro4 transposase


Mass: 38131.738 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: A TEV protease cleavage site was added at the N-terminus (cleaved off) and a 6xHis tag was added at the C-terminus.
Source: (gene. exp.) Citrobacter rodentium ICC168 (bacteria)
Gene: ROD_00481, ROD_11331, ROD_12161, ROD_12961, ROD_23011, ROD_24951, ROD_28391, ROD_30291, ROD_33031, ROD_34671, ROD_35971, ROD_37371, ROD_39361
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Rosetta 2 (DE3) / References: UniProt: D2TGM5
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
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

ComponentName: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex / Type: COMPLEX
Details: ISCro4 tetramer in complex with in vitro transcribed TBL and DBL RNAs and chemically synthesized tDNA and dDNA
Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.263886 MDa / Experimental value: NO
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: Buffer pH = 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethaneTris-HCl1
2500 mMSodium ChlorideNaCl1
35 mMMagnesium ChlorideMgCl21
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA current / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot force:0 Blot time: 3.5 s

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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 defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 66.802 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7particle selection
2EPUimage acquisition
4cryoSPARC4.7CTF correction
7Cootmodel fitting
9cryoSPARC4.7initial Euler assignment
10cryoSPARC4.7final Euler assignment
12cryoSPARC4.73D reconstruction
19PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 268987 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
18WT6

8wt6

A8WT6A1PDBexperimental model
2AlphaFoldin silico model
RefinementHighest resolution: 2.81 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414917
ELECTRON MICROSCOPYf_angle_d0.53921058
ELECTRON MICROSCOPYf_dihedral_angle_d22.4524092
ELECTRON MICROSCOPYf_chiral_restr0.0382433
ELECTRON MICROSCOPYf_plane_restr0.0041959

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