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- PDB-8rc5: Complex between the RecA-like Sak4 SSAP and the SaPI2 Stl master ... -

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Basic information

Entry
Database: PDB / ID: 8rc5
TitleComplex between the RecA-like Sak4 SSAP and the SaPI2 Stl master regulator
Components
  • Helix-turn-helix XRE family protein
  • ORF016
KeywordsRECOMBINATION / Annealase / SSAP / Single Strand Annealing / Single Strand Binding / Recombineering / SaPI / Bacteriophage / Staphylococcal / Complex / SaPI induction / SaPI2 / Mobile Genetic Element / MGE / PICI / Phage-Inducible Chromosomal Island / C18 / Ring
Function / homology
Function and homology information


AAA domain / Cro/C1-type HTH DNA-binding domain / Helix-turn-helix XRE-family like proteins / Cro/C1-type helix-turn-helix domain / Lambda repressor-like, DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Helix-turn-helix XRE family protein / ORF016
Similarity search - Component
Biological speciesStaphylococcus phage 52A (virus)
Staphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsDebiasi-Anders, G. / Mir-Sanchis, I.
Funding support Sweden, 1items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nat Commun / Year: 2025
Title: Phage parasites targeting phage homologous recombinases provide antiviral immunity.
Authors: Gianluca Debiasi-Anders / Cuncun Qiao / Amrita Salim / Na Li / Ignacio Mir-Sanchis /
Abstract: Bacteria often carry multiple genes encoding anti-phage defense systems, clustered in defense islands and phage satellites. Various unrelated anti-phage defense systems target phage-encoded ...Bacteria often carry multiple genes encoding anti-phage defense systems, clustered in defense islands and phage satellites. Various unrelated anti-phage defense systems target phage-encoded homologous recombinases (HRs) through unclear mechanisms. Here, we show that the phage satellite SaPI2, which does not encode orthodox anti-phage defense systems, provides antiviral immunity mediated by Stl2, the SaPI2-encoded transcriptional repressor. Stl2 targets and inhibits phage-encoded HRs, including Sak and Sak4, two HRs from the Rad52-like and Rad51-like superfamilies. Remarkably, apo Stl2 forms a collar of dimers oligomerizing as closed rings and as filaments, mimicking the quaternary structure of its targets. Stl2 decorates both Sak rings and Sak4 filaments. The oligomerization of Stl2 as a collar of dimers is necessary for its inhibitory activity both in vitro and in vivo. Our results shed light on the mechanisms underlying antiviral immunity against phages carrying divergent HRs.
History
DepositionDec 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data 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
1A: ORF016
2A: Helix-turn-helix XRE family protein
3A: Helix-turn-helix XRE family protein
4A: ORF016
1B: ORF016
2B: Helix-turn-helix XRE family protein
3B: Helix-turn-helix XRE family protein
4B: ORF016
1C: ORF016
2C: Helix-turn-helix XRE family protein
3C: Helix-turn-helix XRE family protein
4C: ORF016
1D: ORF016
2D: Helix-turn-helix XRE family protein
3D: Helix-turn-helix XRE family protein
4D: ORF016
1E: ORF016
2E: Helix-turn-helix XRE family protein
3E: Helix-turn-helix XRE family protein
4E: ORF016
1F: ORF016
2F: Helix-turn-helix XRE family protein
3F: Helix-turn-helix XRE family protein
4F: ORF016
1G: ORF016
2G: Helix-turn-helix XRE family protein
3G: Helix-turn-helix XRE family protein
4G: ORF016
1H: ORF016
2H: Helix-turn-helix XRE family protein
3H: Helix-turn-helix XRE family protein
4H: ORF016
1I: ORF016
2I: Helix-turn-helix XRE family protein
3I: Helix-turn-helix XRE family protein
4I: ORF016
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,067,55052
Polymers1,059,17836
Non-polymers8,37216
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
ORF016


Mass: 31653.682 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus phage 52A (virus) / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q4ZAS5
#2: Protein
Helix-turn-helix XRE family protein


Mass: 27189.562 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Strain: RN3984 / Gene: stl / Plasmid: pET21a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0FIL5
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
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: Complex between Sak4 SSAP from staphylococcal phage 52A and the SaPI2 Stl master regulator
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
Details: 100mM NaCl, 20mM Tris-HCl pH 7.0, 1mM DTT and 2mM ATPyS. No metal was added.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium ChlorideNaCl1
220 mMTrisTris1
31 mMDTTDTT1
42 mMATPySATPyS1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1301

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2EPU2.13.0image acquisition
4cryoSPARC4.2.1CTF correctionCTF correction was applied on-the-fly during data collection with CryoSPARC Live.
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.13D reconstruction
CTF correctionDetails: CTF correction was applied on-the-fly during data collection with CryoSPARC Live.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141863
Details: Symmetry-expanded to D1, doubling the particle stack, from 141863 to 283726 particles.
Symmetry type: POINT

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