+Open data
-Basic information
Entry | Database: PDB / ID: 6w5c | ||||||
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Title | Cryo-EM structure of Cas12i(E894A)-crRNA-dsDNA complex | ||||||
Components |
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Keywords | HYDROLASE/DNA/RNA / CRISPR / HYDROLASE-DNA-RNA complex | ||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10) Function and homology information | ||||||
Biological species | Lachnospiraceae bacterium ND2006 (bacteria) Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
Authors | Chang, L. / Li, Z. / Zhang, H. | ||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2020 Title: Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease. Authors: Heng Zhang / Zhuang Li / Renjian Xiao / Leifu Chang / Abstract: Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially ...Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA-DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6w5c.cif.gz | 243.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6w5c.ent.gz | 184.6 KB | Display | PDB format |
PDBx/mmJSON format | 6w5c.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6w5c_validation.pdf.gz | 802.1 KB | Display | wwPDB validaton report |
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Full document | 6w5c_full_validation.pdf.gz | 814.9 KB | Display | |
Data in XML | 6w5c_validation.xml.gz | 33.7 KB | Display | |
Data in CIF | 6w5c_validation.cif.gz | 51.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/w5/6w5c ftp://data.pdbj.org/pub/pdb/validation_reports/w5/6w5c | HTTPS FTP |
-Related structure data
Related structure data | 21541MC 6w62C 6w64C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 125722.008 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria) Production host: Escherichia coli BL21(DE3) (bacteria) |
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#2: DNA chain | Mass: 11083.159 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria) Production host: Escherichia coli BL21(DE3) (bacteria) |
#3: DNA chain | Mass: 2970.954 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria) Production host: Escherichia coli BL21(DE3) (bacteria) |
#4: DNA chain | Mass: 2788.862 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria) Production host: Escherichia coli BL21(DE3) (bacteria) |
#5: RNA chain | Mass: 18859.102 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) |
-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: CRISPR-Cas complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Lachnospiraceae bacterium ND2006 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: unspecified |
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: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 35 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.1_3865: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 731231 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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