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Yorodumi- PDB-8h67: type I-B Cascade bound to a PAM-containing dsDNA target at 3.8 an... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8h67 | |||||||||
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Title | type I-B Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution. | |||||||||
Components |
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Keywords | RNA BINDING PROTEIN / Type I-B / CRISPR-Cas / Cascade | |||||||||
Function / homology | Function and homology information | |||||||||
Biological species | Synechocystis sp. PCC 6714 (bacteria) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Xiao, Y. / Lu, M. / Yu, C. / Zhang, Y. | |||||||||
Funding support | China, 2items
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Citation | Journal: Nat Commun / Year: 2024 Title: Structure and genome editing of type I-B CRISPR-Cas. Authors: Meiling Lu / Chenlin Yu / Yuwen Zhang / Wenjun Ju / Zhi Ye / Chenyang Hua / Jinze Mao / Chunyi Hu / Zhenhuang Yang / Yibei Xiao / Abstract: Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune ...Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3 T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8h67.cif.gz | 563.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8h67.ent.gz | 461.4 KB | Display | PDB format |
PDBx/mmJSON format | 8h67.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8h67_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8h67_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8h67_validation.xml.gz | 98.1 KB | Display | |
Data in CIF | 8h67_validation.cif.gz | 145.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/h6/8h67 ftp://data.pdbj.org/pub/pdb/validation_reports/h6/8h67 | HTTPS FTP |
-Related structure data
Related structure data | 34495MC 8ip0C 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
-RNA chain , 1 types, 1 molecules B
#1: RNA chain | Mass: 22646.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: GenBank: 662706368 |
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-DNA chain , 2 types, 2 molecules CD
#2: DNA chain | Mass: 3390.262 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#3: DNA chain | Mass: 2705.797 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-CRISPR associated protein ... , 4 types, 12 molecules AEFGHJKILMNO
#4: Protein | Mass: 26592.424 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50520 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A068N1Y0 | ||||
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#5: Protein | Mass: 33789.074 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50510 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A068N458 #6: Protein | | Mass: 69287.305 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50500 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A068N831 #7: Protein | Mass: 14538.500 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714 / Gene: D082_50500 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A068N831 |
-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: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution. Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Synechocystis sp. PCC 6714 (bacteria) |
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: 2100 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33011 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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