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- PDB-9y66: attLsym bound serine integrase complex in the dimeric state -

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Basic information

Entry
Database: PDB / ID: 9y66
TitleattLsym bound serine integrase complex in the dimeric state
Components
  • (attLsym) x 2
  • Site-specific recombinase
KeywordsRECOMBINATION / Site-specific DNA Recombinase / Large serine integrase / DNA binding domains / Recombinase / Resolvase / Zinc ribbon recombinase
Function / homology
Function and homology information


DNA strand exchange activity / DNA binding
Similarity search - Function
Recombinase / DNA-binding recombinase domain / DNA-binding recombinase domain superfamily / DNA-binding recombinase domain profile. / : / Resolvase/invertase-type recombinase catalytic domain profile. / Resolvase, N-terminal catalytic domain / Resolvase-like, N-terminal catalytic domain superfamily / Resolvase, N terminal domain / Resolvase, N terminal domain
Similarity search - Domain/homology
DNA / DNA (> 10) / Site-specific recombinase
Similarity search - Component
Biological speciesSpbetavirus SPbeta
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsShin, H. / Pigli, Y. / Pena Reyes, T. / Fuller, J.R. / Olorunniji, F.J. / Rice, P.A.
Funding support United States, United Kingdom, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)2223480 United States
UK Research and Innovation (UKRI)2223480 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)2223480 United Kingdom
CitationJournal: bioRxiv / Year: 2025
Title: Structural basis of directionality control in large serine integrases.
Authors: Heewhan Shin / Ying Pigli / Tania Peña Reyes / James R Fuller / Femi J Olorunniji / Phoebe A Rice /
Abstract: Large serine integrases (LSIs) catalyze unidirectional site-specific DNA recombination reactions, yet those reactions are reversed by the presence of a cognate recombination directionality factor ...Large serine integrases (LSIs) catalyze unidirectional site-specific DNA recombination reactions, yet those reactions are reversed by the presence of a cognate recombination directionality factor (RDF). Mechanistic understanding of directionality control has been hampered by a lack of structural information. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of six SPbeta integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at 4.16-7.18Å resolution. Our findings reveal how RDF-mediated repositioning of an integrase subdomain (1) dictates which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination and (2) dictates which product complexes will be conformationally locked, preventing the back reaction. These mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.
History
DepositionSep 8, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 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: Site-specific recombinase
C: Site-specific recombinase
E: attLsym
F: attLsym
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,4186
Polymers165,2874
Non-polymers1312
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 Site-specific recombinase


Mass: 63232.066 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spbetavirus SPbeta / Gene: yokA
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: O64015
#2: DNA chain attLsym


Mass: 19436.590 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Spbetavirus SPbeta
#3: DNA chain attLsym


Mass: 19386.469 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Spbetavirus SPbeta
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / 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

ComponentName: Dimeric complex of attLsym bound large serine integrases
Type: COMPLEX
Details: DNAs synthesized from Integrated DNA Technologies (IDT)
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Spbetavirus SPbeta
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8 / Details: 20mM Tris-HCL pH 8, 100mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
220 mMTrisC4H11NO31
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291.15 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 defocus max: 1900 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2PHENIX1.21.1_5286:model refinement
10cryoSPARC4.6.0initial Euler assignment
13cryoSPARC4.6.03D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 263209 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511713
ELECTRON MICROSCOPYf_angle_d0.71616295
ELECTRON MICROSCOPYf_dihedral_angle_d19.8154921
ELECTRON MICROSCOPYf_chiral_restr0.0561807
ELECTRON MICROSCOPYf_plane_restr0.0131659

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