+Open data
-Basic information
Entry | Database: PDB / ID: 8wm4 | ||||||||||||
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Title | Cryo-EM structure of DiCas7-11 in complex with crRNA | ||||||||||||
Components |
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Keywords | IMMUNE SYSTEM / CRISPR-Cas / Gene editing / TPR-CHAT | ||||||||||||
Function / homology | CRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA / RNA (> 10) / CRISPR-associated RAMP family protein Function and homology information | ||||||||||||
Biological species | Desulfonema ishimotonii (bacteria) Escherichia coli (E. coli) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å | ||||||||||||
Authors | Ma, H.Y. / Tang, X.D. | ||||||||||||
Funding support | China, 3items
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Citation | Journal: Signal Transduct Target Ther / Year: 2024 Title: Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT. Authors: Tian Hong / Qinghua Luo / Haiyun Ma / Xin Wang / Xinqiong Li / Chongrong Shen / Jie Pang / Yan Wang / Yuejia Chen / Changbin Zhang / Zhaoming Su / Haohao Dong / Xiaodi Tang / Abstract: CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as ...CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHAT). Our work demonstrates that DiTPR-CHAT can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8wm4.cif.gz | 303.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8wm4.ent.gz | 236.9 KB | Display | PDB format |
PDBx/mmJSON format | 8wm4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8wm4_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8wm4_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8wm4_validation.xml.gz | 52.4 KB | Display | |
Data in CIF | 8wm4_validation.cif.gz | 78.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wm/8wm4 ftp://data.pdbj.org/pub/pdb/validation_reports/wm/8wm4 | HTTPS FTP |
-Related structure data
Related structure data | 37640MC 8wmcC 8wmiC 8wmlC 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: 183933.000 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Gene: DENIS_1082 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A401FT36 | ||
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#2: RNA chain | Mass: 12134.133 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) | ||
#3: Chemical | ChemComp-ZN / Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: CELL / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Binary complex of Cas7-11 and crRNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: NITROGEN / Humidity: 100 % |
-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 magnification: 165000 X / Nominal defocus max: 3035 nm / Nominal defocus min: 980 nm / Cs: 2.7 mm |
Image recording | Electron dose: 61.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 216156 / Symmetry type: POINT | ||||||||||||||||||||||||
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