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- PDB-8ifk: Cryo-EM structure of monomeric SPARTA gRNA-ssDNA target complex -

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Basic information

Entry
Database: PDB / ID: 8ifk
TitleCryo-EM structure of monomeric SPARTA gRNA-ssDNA target complex
Components
  • Piwi domain-containing protein
  • TIR domain-containing protein
  • guide RNA
  • target ssDNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA COMPLEX
Function / homology
Function and homology information


nucleic acid binding / signal transduction / metal ion binding
Similarity search - Function
TIR domain / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesThermoflavifilum thermophilum (bacteria)
Escherichia coli 'BL21-GoldpLysS AG'
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsZhang, J.T. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Chem Biol / Year: 2024
Title: Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system.
Authors: Jun-Tao Zhang / Xin-Yang Wei / Ning Cui / Ruilin Tian / Ning Jia /
Abstract: Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: ...Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: the short prokaryotic Ago/TIR-APAZ (SPARTA) immune system triggers cell death by degrading NAD in response to invading plasmids, but its molecular mechanisms remain unknown. Here we used cryo-electron microscopy to determine the structures of inactive monomeric and active tetrameric Crenotalea thermophila SPARTA complexes, revealing mechanisms underlying SPARTA assembly, RNA-guided recognition of target single-stranded DNA (ssDNA) and subsequent SPARTA tetramerization, as well as tetramerization-dependent NADase activation. The small RNA guides Ago to recognize its ssDNA target, inducing SPARTA tetramerization via both Ago- and TIR-mediated interactions and resulting in a two-stranded, parallel, head-to-tail TIR rearrangement primed for NAD hydrolysis. Our findings thus identify the molecular basis for target ssDNA-mediated SPARTA activation, which will facilitate the development of SPARTA-based biotechnological tools.
History
DepositionFeb 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TIR domain-containing protein
B: Piwi domain-containing protein
C: guide RNA
D: target ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,9265
Polymers125,9024
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein TIR domain-containing protein


Mass: 53256.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1670
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A1I7NFG5
#2: Protein Piwi domain-containing protein / CrtAgo


Mass: 58304.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1671
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A1I7NFD7
#3: RNA chain guide RNA


Mass: 6658.989 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: DNA chain target ssDNA


Mass: 7680.997 Da / Num. of mol.: 1 / 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.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: CrtSPARTA-gRNA-tDNA ternary complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Thermoflavifilum thermophilum (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 / Cs: 2.7 mm / 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

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 615987 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0097899
ELECTRON MICROSCOPYf_angle_d0.94910854
ELECTRON MICROSCOPYf_dihedral_angle_d14.4781325
ELECTRON MICROSCOPYf_chiral_restr0.0421193
ELECTRON MICROSCOPYf_plane_restr0.0061219

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