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- PDB-8ifl: Cryo-EM structure of tetrameric SPARTA gRNA-ssDNA target complex ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8ifl | ||||||
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Title | Cryo-EM structure of tetrameric SPARTA gRNA-ssDNA target complex in state 1 | ||||||
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![]() | RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA COMPLEX | ||||||
Function / homology | ![]() | ||||||
Biological species | ![]() Escherichia coli 'BL21-GoldpLysS AG' | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å | ||||||
![]() | Zhang, J.T. / Jia, N. | ||||||
Funding support | 1items
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 775.4 KB | Display | ![]() |
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PDB format | ![]() | 621.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.7 MB | Display | ![]() |
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Full document | ![]() | 1.8 MB | Display | |
Data in XML | ![]() | 116.2 KB | Display | |
Data in CIF | ![]() | 176.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 35420MC ![]() 8ifkC ![]() 8ifmC ![]() 8k34C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 53256.734 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: SAMN05660895_1670 Production host: ![]() ![]() References: UniProt: A0A1I7NFG5 #2: Protein | Mass: 58304.848 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: SAMN05660895_1671 Production host: ![]() ![]() References: UniProt: A0A1I7NFD7 #3: RNA chain | Mass: 6658.989 Da / Num. of mol.: 4 / Source method: obtained synthetically Source: (synth.) ![]() ![]() #4: DNA chain | Mass: 7680.997 Da / Num. of mol.: 4 / Source method: obtained synthetically Source: (synth.) ![]() ![]() #5: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: CrtSPARTA tetramer complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
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Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / 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) |
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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: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171346 / Symmetry type: POINT | ||||||||||||||||||||||||
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