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- PDB-8pq8: Sak Single Strand Annealing Protein from Staphylococcal Bacteriop... -

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Basic information

Entry
Database: PDB / ID: 8pq8
TitleSak Single Strand Annealing Protein from Staphylococcal Bacteriophage 80a - dCTD
ComponentsTopoisomerase
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 / homologySingle-strand annealing protein SAK3 / Sak Single Strand Annealing Protein / isomerase activity / Topoisomerase
Function and homology information
Biological speciesStaphylococcus phage 80alpha (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 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
DepositionJul 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Topoisomerase


Theoretical massNumber of molelcules
Total (without water)19,4181
Polymers19,4181
Non-polymers00
Water1629
1
A: Topoisomerase
x 18


Theoretical massNumber of molelcules
Total (without water)349,51718
Polymers349,51718
Non-polymers00
Water32418
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation17

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Components

#1: Protein Topoisomerase / Sak Single-Strand Annealing Protein


Mass: 19417.621 Da / Num. of mol.: 1 / Mutation: Truncation of CTD at N142
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus phage 80alpha (virus) / Cell (production host): NA / Organ (production host): NA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Tissue (production host): NA / References: UniProt: C8CGY8
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O
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: 18-mer ring cryoEM structure of the 80a-Sak NTD in apo state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.46 MDa / Experimental value: YES
Source (natural)Organism: Staphylococcus phage 80alpha (virus) / Cellular location: NA / Organ: NA / Organelle: NA / Tissue: NA
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8.1 / Details: 20mM Tris-HCl pH 7.6, 100mM NaCl, 1mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl pH 7.6Tris-HCl1
2100 mMSodium ChlorideNaCl1
31 mMDithiothreitolDTT1
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: 2900 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.99 sec. / Electron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4344

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2EPU2.13.0image acquisition
4cryoSPARC4.2.1CTF correctionCTF correct on-the-fly with cryoSPARC Live
7UCSF ChimeraX1.4model fitting
9PHENIX1.20.1model refinement
10Coot0.9.8.4 ELmodel refinement
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
14cryoSPARC4.2.13D reconstructionLocal refinement job after symmetry expansion
CTF correctionDetails: CTF correction was applied on-the-fly during data collection with CryoSPARC Live.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C18 (18 fold cyclic)
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75703
Details: Symmetry expanded to C18, multiplying the stack by 18, from 75703 to 1365300 particles.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Fitting of the truncated AlphaFold prediction was done with ChimeraX and then subjected to real space refinement in PHENIX. The output was then repeatedly adjusted with Coot and verified in PHENIX.
Atomic model buildingChain residue range: 1-141
Details: AlphaFold prediction was done locally. CTD residues of the full-length AlphaFold prediction were deleted before fiting with ChimeraX.
Source name: AlphaFold / Type: in silico model

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