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- EMDB-55572: Cryo-EM structure of ISCro4-DBL-TBL-tDNA-dDNA synaptic complex -

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Basic information

Entry
Database: EMDB / ID: EMD-55572
TitleCryo-EM structure of ISCro4-DBL-TBL-tDNA-dDNA synaptic complex
Map data
Sample
  • Complex: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex
    • Protein or peptide: ISCro4 transposase
    • RNA: Target-binding loop (TBL) RNA
    • RNA: Donor-binding loop (DBL) RNA
    • DNA: target DNA (tRNA) top strand
    • DNA: target DNA (tDNA) bottom strand
    • DNA: donor DNA (dDNA) top strand
    • DNA: donor DNA (dDNA) bottom strand
  • Ligand: MAGNESIUM ION
KeywordsNucleic acid interaction / RNA binding / DNA binding / tetramer / recombination / recombinase
Function / homologyTransposase, IS111A/IS1328/IS1533, N-terminal / : / Transposase / Transposase, IS116/IS110/IS902 / Transposase IS116/IS110/IS902 family / transposase activity / DNA transposition / DNA binding / ISCro4 transposase
Function and homology information
Biological speciesCitrobacter rodentium ICC168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsFernandez Carrera J / Pelea O / Gerecke SE / Chanez C / Jinek M
Funding supportEuropean Union, Switzerland, 2 items
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, 2025-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateFeb 18, 2026-
Current statusFeb 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55572.png

Downloads & links

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Map

FileDownload / File: emd_55572.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 400 pix.
= 260. Å		0.65 Å/pix.
x 400 pix.
= 260. Å		0.65 Å/pix.
x 400 pix.
= 260. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0349
Minimum - Maximum-0.052142285 - 0.15215085
Average (Standard dev.)0.00019010143 (±0.0044270772)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_55572_msk_1.map
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AxesZYX

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Additional map: Sharpened map

Fileemd_55572_additional_1.map
AnnotationSharpened map
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Half map: Half map B

Fileemd_55572_half_map_1.map
AnnotationHalf map B
Projections & Slices
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Half map: Half map A

Fileemd_55572_half_map_2.map
AnnotationHalf map A
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Sample components

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Entire : ISCro4-DBL-TBL-tDNA-dDNA synaptic complex

EntireName: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex
Components
  • Complex: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex
    • Protein or peptide: ISCro4 transposase
    • RNA: Target-binding loop (TBL) RNA
    • RNA: Donor-binding loop (DBL) RNA
    • DNA: target DNA (tRNA) top strand
    • DNA: target DNA (tDNA) bottom strand
    • DNA: donor DNA (dDNA) top strand
    • DNA: donor DNA (dDNA) bottom strand
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex

SupramoleculeName: ISCro4-DBL-TBL-tDNA-dDNA synaptic complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: ISCro4 tetramer in complex with in vitro transcribed TBL and DBL RNAs and chemically synthesized tDNA and dDNA
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 263.886 KDa

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Macromolecule #1: ISCro4 transposase

MacromoleculeName: ISCro4 transposase / type: protein_or_peptide / ID: 1
Details: A TEV protease cleavage site was added at the N-terminus (cleaved off) and a 6xHis tag was added at the C-terminus.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 38.131738 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMEQELHF IGIDVSKAKL DVDVLRPDGR HRSKKFANTP KGHDELLRWL SGHRVAPAHI CMEATSTYME DVAAHLSDAG YTVSVINPA LGKAFAQSEG LRSKTDAVDA RMLAEFCRQK RPPAWEAPHP VERALRALVL RHQSLTDMHT QELNRLETAR E VQRPSIDA ...String:
SNAMEQELHF IGIDVSKAKL DVDVLRPDGR HRSKKFANTP KGHDELLRWL SGHRVAPAHI CMEATSTYME DVAAHLSDAG YTVSVINPA LGKAFAQSEG LRSKTDAVDA RMLAEFCRQK RPPAWEAPHP VERALRALVL RHQSLTDMHT QELNRLETAR E VQRPSIDA HLLWLHAELK RIEKQIKDLT DDDPDMKHRR KLLESIPGIG EKTSAVLLAY TGLKERFTHA RQFAAFAGLT PR RYESGSS VNRASRMSKA GHASLRRALY MPAMVAVSKT EWGRAFRDRL AGNGKKGKVI IGAMMRKLAQ VAYGVLKSGV PFD ASRHNP VAASSGSLEH HHHHH

UniProtKB: ISCro4 transposase

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

MacromoleculeName: Target-binding loop (TBL) RNA / type: rna / ID: 2
Details: GGG added the 5'-end and UUUU appended at the 3'-end to facilitate in vitro transcription
Number of copies: 1
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 35.682082 KDa
SequenceString:
GGGAGUGCAG GGAGAACCGG CCAGUUCUCU CUGCCAUGCG GUCCGCAUGC CGUAUCAGGC CUCAGGCUAA UAACCUGUGA CGUAGAUUU AUGCAGCGGA CCGCCGUUUU UU

GENBANK: GENBANK: FN543502.1

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Macromolecule #3: Donor-binding loop (DBL) RNA

MacromoleculeName: Donor-binding loop (DBL) RNA / type: rna / ID: 3
Details: GGG added the 5'-end and UUUUUU appended at the 3'-end to facilitate in vitro transcription
Number of copies: 1
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 25.278945 KDa
SequenceString:
GGGCAAGUGA CAAACCGGAC AGUAUCAUGG ACCGGUUUUC CCGGUAAUCC GCAUUCACAA GGCUGGUCUC ACUUUUUUU

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Macromolecule #4: target DNA (tRNA) top strand

MacromoleculeName: target DNA (tRNA) top strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 11.616454 KDa
SequenceString:
(DG)(DC)(DC)(DG)(DG)(DG)(DT)(DA)(DA)(DT) (DA)(DC)(DC)(DC)(DT)(DT)(DA)(DT)(DC)(DA) (DG)(DG)(DC)(DC)(DT)(DA)(DC)(DG)(DT) (DC)(DA)(DC)(DA)(DG)(DC)(DT)(DC)(DG)

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Macromolecule #5: target DNA (tDNA) bottom strand

MacromoleculeName: target DNA (tDNA) bottom strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 11.776551 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DG)(DA) (DC)(DG)(DT)(DA)(DG)(DG)(DC)(DC)(DT)(DG) (DA)(DT)(DA)(DA)(DG)(DG)(DG)(DT)(DA) (DT)(DT)(DA)(DC)(DC)(DC)(DG)(DG)(DC)

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Macromolecule #6: donor DNA (dDNA) top strand

MacromoleculeName: donor DNA (dDNA) top strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 13.562724 KDa
SequenceString:
(DT)(DG)(DC)(DA)(DG)(DG)(DC)(DG)(DA)(DT) (DA)(DA)(DC)(DA)(DG)(DT)(DA)(DT)(DC)(DT) (DT)(DG)(DT)(DA)(DT)(DT)(DA)(DT)(DC) (DC)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DG)(DC) (DA) (DG)(DA)(DG)(DA)

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Macromolecule #7: donor DNA (dDNA) bottom strand

MacromoleculeName: donor DNA (dDNA) bottom strand / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Citrobacter rodentium ICC168 (bacteria)
Molecular weightTheoretical: 13.531716 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DT)(DG)(DC)(DA)(DC)(DT) (DG)(DA)(DA)(DG)(DG)(DG)(DA)(DT)(DA)(DA) (DT)(DA)(DC)(DA)(DA)(DG)(DA)(DT)(DA) (DC)(DT)(DG)(DT)(DT)(DA)(DT)(DC)(DG)(DC) (DC) (DT)(DG)(DC)(DA)

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / 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

Concentration1.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HCltris(hydroxymethyl)aminomethane
500.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride

Details: Buffer pH = 8.0
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.039 kPa / Details: 15 mA current
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force:0 Blot time: 3.5 s.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.802 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.7) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8wt6

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7) / Number images used: 268987
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.7)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4.7)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

8wt6

chain_id: A, source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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