+Open data
-Basic information
Entry | Database: PDB / ID: 7v93 | ||||||
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Title | Cryo-EM structure of the Cas12c2-sgRNA binary complex | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA / cas12c / c2c3 / sgRNA / CRISPR / RNA binding protein-RNA complex | ||||||
Function / homology | RNA / RNA (> 10) / RNA (> 100) Function and homology information | ||||||
Biological species | uncultured archaeon (environmental samples) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||
Authors | Kurihara, N. / Hirano, H. / Tomita, A. / Kobayashi, K. / Kusakizako, T. / Nishizawa, T. / Yamashita, K. / Nishimasu, H. / Nureki, O. | ||||||
Funding support | Japan, 1items
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Citation | Journal: Mol Cell / Year: 2022 Title: Structure of the type V-C CRISPR-Cas effector enzyme. Authors: Nina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi ...Authors: Nina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi Nishimasu / Osamu Nureki / Abstract: RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7v93.cif.gz | 245.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7v93.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7v93.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7v93_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7v93_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7v93_validation.xml.gz | 38 KB | Display | |
Data in CIF | 7v93_validation.cif.gz | 59.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/v9/7v93 ftp://data.pdbj.org/pub/pdb/validation_reports/v9/7v93 | HTTPS FTP |
-Related structure data
Related structure data | 31807MC 7v94C 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: 138348.938 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) uncultured archaeon (environmental samples) Production host: Escherichia coli (E. coli) |
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#2: RNA chain | Mass: 36147.414 Da / Num. of mol.: 1 / Source method: isolated from a natural source Source: (natural) uncultured archaeon (environmental samples) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||
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 |
Image recording | Average exposure time: 5 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software | Name: REFMAC / Version: 5.8.0267 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289394 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: RECIPROCAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3→3 Å / Cor.coef. Fo:Fc: 0.908 / SU B: 13.97 / SU ML: 0.263 / ESU R: 0.428 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 107.322 Å2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: 1 / Total: 8792 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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