+Open data
-Basic information
Entry | Database: PDB / ID: 8i3q | ||||||
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Title | Cryo-EM structure of Cas12g-sgRNA binary complex | ||||||
Components |
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Keywords | RNA BINDING PROTEIN / CRISPR-Cas | ||||||
Function / homology | RNA / RNA (> 10) / RNA (> 100) Function and homology information | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Ouyang, S.Y. / Liu, M.X. / Li, Z.K. | ||||||
Funding support | China, 1items
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Citation | Journal: PLoS Genet / Year: 2023 Title: Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage. Authors: Mengxi Liu / Zekai Li / Jing Chen / Jinying Lin / Qiuhua Lu / Yangmiao Ye / Hongmin Zhang / Bo Zhang / Songying Ouyang / Abstract: Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC ...Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8i3q.cif.gz | 172.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8i3q.ent.gz | 126.8 KB | Display | PDB format |
PDBx/mmJSON format | 8i3q.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/i3/8i3q ftp://data.pdbj.org/pub/pdb/validation_reports/i3/8i3q | HTTPS FTP |
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-Related structure data
Related structure data | 35154MC 8i16C 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: RNA chain | Mass: 44771.703 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
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#2: Protein | Mass: 89847.672 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria) |
#3: Chemical | ChemComp-ZN / |
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: Escherichia coli (E. coli) | ||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | ||||||||||||||||||||||||
Buffer solution | pH: 8.5 | ||||||||||||||||||||||||
Specimen | Conc.: 3 mg/ml / 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: 1800 nm / Nominal defocus min: 1200 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88696 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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