+Open data
-Basic information
Entry | Database: PDB / ID: 8wcs | ||||||
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Title | Cryo-EM structure of Cas13h1-crRNA binary complex | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA COMPLEX | ||||||
Function / homology | RNA / RNA (> 10) Function and homology information | ||||||
Biological species | unidentified (others) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Zhang, C. | ||||||
Funding support | China, 1items
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Citation | Journal: Nucleic Acids Res / Year: 2024 Title: Molecular mechanism for target RNA recognition and cleavage of Cas13h. Authors: Fugen Chen / Chendi Zhang / Jialin Xue / Feng Wang / Zhuang Li / Abstract: RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA ...RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8wcs.cif.gz | 234.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8wcs.ent.gz | 181.4 KB | Display | PDB format |
PDBx/mmJSON format | 8wcs.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8wcs_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8wcs_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8wcs_validation.xml.gz | 42.5 KB | Display | |
Data in CIF | 8wcs_validation.cif.gz | 64.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wc/8wcs ftp://data.pdbj.org/pub/pdb/validation_reports/wc/8wcs | HTTPS FTP |
-Related structure data
Related structure data | 37448MC 8wceC 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: 130957.328 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli) |
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#2: RNA chain | Mass: 21257.666 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: 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 |
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Source (natural) | Organism: unidentified (others) | ||||||||||||||||||||||||
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: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm |
Image recording | Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: NONE | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207575 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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