+Open data
-Basic information
Entry | Database: PDB / ID: 8d4b | ||||||||||||
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Title | Structure of Cas12a2 ternary complex | ||||||||||||
Components |
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Keywords | DNA Binding Protein/RNA / Cas12a2 / CRISPR / Nuclease / DNA Binding Protein-RNA complex | ||||||||||||
Function / homology | Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / DNA binding / RNA / RNA (> 10) / Cas12f1-like TNB domain-containing protein Function and homology information | ||||||||||||
Biological species | Sulfuricurvum sp. PC08-66 (bacteria) synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å | ||||||||||||
Authors | Bravo, J.P.K. / Taylor, D.W. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Nature / Year: 2023 Title: RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2. Authors: Jack P K Bravo / Thomson Hallmark / Bronson Naegle / Chase L Beisel / Ryan N Jackson / David W Taylor / Abstract: Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a ...Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8d4b.cif.gz | 255.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8d4b.ent.gz | 197.3 KB | Display | PDB format |
PDBx/mmJSON format | 8d4b.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8d4b_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8d4b_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8d4b_validation.xml.gz | 43.8 KB | Display | |
Data in CIF | 8d4b_validation.cif.gz | 65.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/d4/8d4b ftp://data.pdbj.org/pub/pdb/validation_reports/d4/8d4b | HTTPS FTP |
-Related structure data
Related structure data | 27180MC 8d49C 8d4aC 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: 143172.797 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Sulfuricurvum sp. PC08-66 (bacteria) / Gene: KU37_03970 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0C2W1L1 |
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#2: RNA chain | Mass: 13151.831 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: RNA chain | Mass: 8934.285 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-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: Cas12a2 ternary complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Sulfuricurvum sp. PC08-66 (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
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: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm |
Image recording | Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192639 / Symmetry type: POINT | ||||||||||||||||||||||||
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