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- PDB-8y80: Cryo-EM structure of the tetrameric SPARSA gRNA-ssDNA complex -

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Basic information

Entry
Database: PDB / ID: 8y80
TitleCryo-EM structure of the tetrameric SPARSA gRNA-ssDNA complex
Components
  • DNA (25-mer)
  • Piwi domain protein
  • RNA (21-mer)
  • Sir2 superfamily protein
KeywordsSTRUCTURAL PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA COMPLEX / STRUCTURAL PROTEIN / STRUCTURAL PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
SIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain protein / Sir2 superfamily protein
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
Escherichia coli 'BL21-GoldpLysS AG'
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsZhang, J.T. / Cui, N. / Wei, X.Y. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system.
Authors: Ning Cui / Jun-Tao Zhang / Zhuolin Li / Xin-Yang Wei / Jie Wang / Ning Jia /
Abstract: Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids ...Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5'-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.
History
DepositionFeb 5, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sir2 superfamily protein
B: Piwi domain protein
C: RNA (21-mer)
D: DNA (25-mer)
E: Sir2 superfamily protein
F: Piwi domain protein
G: RNA (21-mer)
H: DNA (25-mer)
I: Sir2 superfamily protein
J: Piwi domain protein
K: RNA (21-mer)
L: DNA (25-mer)
M: Sir2 superfamily protein
N: Piwi domain protein
O: RNA (21-mer)
P: DNA (25-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)537,42320
Polymers537,32616
Non-polymers974
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
Sir2 superfamily protein


Mass: 66645.922 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1360
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q74DF6
#2: Protein
Piwi domain protein


Mass: 53325.566 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1361
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q74DF5
#3: RNA chain
RNA (21-mer)


Mass: 6651.949 Da / Num. of mol.: 4 / Source method: obtained synthetically
Source: (synth.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: DNA chain
DNA (25-mer)


Mass: 7708.040 Da / Num. of mol.: 4 / Source method: obtained synthetically
Source: (synth.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / 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: Tetrameric SPARSA gRNA-ssDNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36656 / Symmetry type: POINT
RefinementHighest resolution: 3.38 Å

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