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Basic information

Entry
Database: PDB / ID: 9jsp
Titleinactive NbaSPARDA complexes
Components
  • Ago
  • DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')
  • DREN-APAZ
  • RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')
KeywordsRNA BINDING PROTEIN/RNA/DNA / protein / RNA / DNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesNovosphingopyxis baekryungensis DSM 16222 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsZhuang, L.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2025
Title: Target DNA-induced filament formation and nuclease activation of SPARDA complex.
Authors: Feng Wang / Haijiang Xu / Chendi Zhang / Jialin Xue / Zhuang Li /
Abstract: The short Argonaute-based bacterial defense system, SPARDA (Short Prokaryotic Argonaute and DNase/RNase-APAZ), utilizes guide RNA to target invading complementary DNA and exhibits collateral nuclease ...The short Argonaute-based bacterial defense system, SPARDA (Short Prokaryotic Argonaute and DNase/RNase-APAZ), utilizes guide RNA to target invading complementary DNA and exhibits collateral nuclease activity, leading to cell death or dormancy. However, its detailed mechanisms remain poorly understood. In this study, we investigated the SPARDA system from Novosphingopyxis baekryungensis (NbaSPARDA) and discovered an unexpected filament configuration upon target DNA binding, which strongly correlated with collateral nuclease activity. Filament formation and nuclease activation require a guide-target heteroduplex of sufficient length with perfect complementarity at the central region. A series of cryo-EM structures of NbaSPARDA complexes, loaded with guide RNA, target DNA of varying lengths, and substrate ssDNA, were determined at ~3.0 Å resolution. Structural analyses indicated that guide RNA binding induces dimerization of the NbaSPARDA complex, while target DNA engagement disrupts this dimerization. Further propagation of the guide-target heteroduplex triggers filament formation through a checkpoint mechanism. The NbaSPARDA filament consists of a backbone formed by interlocking short Argonaute proteins, with an inner layer composed of DREN nuclease domains. Filament formation leads to tetramerization of the monomeric DREN nuclease domain, activating its collateral nuclease activity against environmental nucleic acids - a feature leveraged for molecular diagnostics. For bacteria heterologously expressing the NbaSPARDA system, defense against invading bacteriophages and plasmids relies on filament formation. Collectively, these findings illustrate the detailed working mechanism of the NbaSPARDA complex and highlight the importance of its filament formation in host defense.
History
DepositionSep 30, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 2, 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: Ago
B: DREN-APAZ
G: RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')
T: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,3255
Polymers115,3014
Non-polymers241
Water00
1


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

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Components

#1: Protein Ago


Mass: 54492.809 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein DREN-APAZ


Mass: 50419.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#3: RNA chain RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')


Mass: 6390.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#4: DNA chain DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')


Mass: 3997.607 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / 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: pro-RNA-13DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213852 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046046
ELECTRON MICROSCOPYf_angle_d0.8918277
ELECTRON MICROSCOPYf_dihedral_angle_d11.585980
ELECTRON MICROSCOPYf_chiral_restr0.049897
ELECTRON MICROSCOPYf_plane_restr0.009990

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