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- PDB-9why: Cryo-EM structure of the IS621 recombinase in complex with bridge... -

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Basic information

Entry
Database: PDB / ID: 9why
TitleCryo-EM structure of the IS621 recombinase in complex with bridge RNA, left-half DNA, and right-half DNA in the post-strand exchange state
Components
  • IS621 transposase
  • LH, bottom strand
  • LH/RH, top strand
  • RH, bottom strand
  • RH/LH, top strand
  • bridge RNA
KeywordsDNA BINDING PROTEIN / Holliday junction / RNA dependent recombinase / RECOMBINATION-DNA-RNA complex
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) / IS621 transposase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsHiraizumi, M. / Tsujimoto, E. / Shiojiri, N. / Nagahata, N. / Yamashita, K. / Nishimasu, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR23B6 Japan
Other privateInamori Foundation
CitationJournal: Nature / Year: 2026
Title: Structural mechanism governing the directionality of bridge recombination.
Authors: Masahiro Hiraizumi / Januka S Athukoralage / Nicholas T Perry / Eisuke Tsujimoto / Nami Shiojiri / Naoto Nagahata / Lauren Lee / Gwanggyu Sun / Matthew G Durrant / Sita S Chandrasekaran / ...Authors: Masahiro Hiraizumi / Januka S Athukoralage / Nicholas T Perry / Eisuke Tsujimoto / Nami Shiojiri / Naoto Nagahata / Lauren Lee / Gwanggyu Sun / Matthew G Durrant / Sita S Chandrasekaran / Silvana Konermann / Keitaro Yamashita / Patrick D Hsu / Hiroshi Nishimasu /
Abstract: Bridge recombinases from the IS110 family of transposons, such as IS621, associate with a bridge RNA (bRNA) to mediate programmable recombination between donor DNA and target DNA. Although insertion ...Bridge recombinases from the IS110 family of transposons, such as IS621, associate with a bridge RNA (bRNA) to mediate programmable recombination between donor DNA and target DNA. Although insertion is mediated by the recombinase-bRNA complex, it remains unknown how IS621 elements are excised from host genomes to form the circular DNA intermediates required for transposition. Here we show that bRNA is weakly expressed from IS621 loci in the Escherichia coli genome and that the IS621 recombinase-bRNA complex mediates excision less efficiently than insertion. Furthermore, we present the cryo-electron microscopy structures of the IS621 recombinase-bRNA complex bound to excision DNA substrates, providing mechanistic insights into the excision reaction. Similar to the previously reported donor- and target-bound insertion complex, the excision complex comprises two recombinase dimers, each accommodating the target- and donor-binding loops of the bRNA. However, DNA recognition differs notably between the two complexes. Although the donor and target DNAs form a bent U-shape during insertion, the excision substrates adopt linear conformations and bind across both bRNA loops, forming an X-shaped structure. This geometry reduces the efficiency of top-strand exchange and contributes to the naturally observed bias favouring insertion over excision. Despite these differences, the efficiencies of both reactions are similarly modulated by base pairing between specific dinucleotides in the bRNA, termed handshake guides, and the top strands of the DNA substrates. Overall, this study provides mechanistic insights into the complete IS110 transposition cycle and facilitates the optimal design of programmable bridge-editing applications.
History
DepositionAug 27, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 12, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Aug 26, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Aug 26, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: IS621 transposase
B: IS621 transposase
C: IS621 transposase
D: IS621 transposase
E: bridge RNA
F: bridge RNA
G: LH/RH, top strand
H: LH, bottom strand
I: RH/LH, top strand
J: RH, bottom strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,78812
Polymers312,74010
Non-polymers492
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 4 types, 4 molecules GHIJ

#3: DNA chain LH/RH, top strand


Mass: 15032.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain LH, bottom strand


Mass: 11809.589 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#5: DNA chain RH/LH, top strand


Mass: 10055.483 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain RH, bottom strand


Mass: 13562.671 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 6 molecules ABCDEF

#1: Protein
IS621 transposase


Mass: 36735.355 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: O3K_03330, O3K_03970, O3K_05990, O3K_07835, O3K_09380, O3K_16710, O3K_17245, O3K_20600, O3K_22425
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0E0Y1P1
#2: RNA chain bridge RNA


Mass: 57668.918 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: cell-free synthesis / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 6 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Cryo-EM structure of the IS621 recombinase in complex with bridge RNA, left-half DNA, and right-half DNA in the post-strand exchange state
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
9Servalcatmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 515470 / Symmetry type: POINT

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