+Open data
-Basic information
Entry | Database: PDB / ID: 8k34 | ||||||
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Title | Cryo-EM structure of SPARTA gRNA binary complex | ||||||
Components |
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Keywords | STRUCTURAL PROTEIN/RNA / STRUCTURAL PROTEIN-RNA COMPLEX | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Thermoflavifilum thermophilum (bacteria) Escherichia coli 'BL21-GoldpLysS AG' | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å | ||||||
Authors | Zhang, J.T. / Jia, N. | ||||||
Funding support | 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8k34.cif.gz | 193 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8k34.ent.gz | 148.2 KB | Display | PDB format |
PDBx/mmJSON format | 8k34.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/k3/8k34 ftp://data.pdbj.org/pub/pdb/validation_reports/k3/8k34 | HTTPS FTP |
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-Related structure data
Related structure data | 36843MC 8ifkC 8iflC 8ifmC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: 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 |
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#2: Protein | 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 | Mass: 6658.989 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
#4: Chemical | ChemComp-MG / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: CrtSPARTA-gRNA binary complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Source (natural) | Organism: Thermoflavifilum thermophilum (bacteria) |
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software | Name: PHENIX / Version: 1.20.1_4487: / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500000 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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