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- PDB-8ip0: Cryo-EM structure of type I-B Cascade bound to a PAM-containing d... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8ip0 | |||||||||
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Title | Cryo-EM structure of type I-B Cascade bound to a PAM-containing dsDNA target at 3.6 angstrom resolution | |||||||||
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![]() | RNA BINDING PROTEIN/RNA/DNA / Type I-B / CRISPR-Cas / Cascade / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | ![]() | |||||||||
Biological species | ![]() ![]() synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
![]() | Xiao, Y. / Lu, M. / Yu, C. / Zhang, Y. | |||||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 586.8 KB | Display | ![]() |
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PDB format | ![]() | 476.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 474.5 KB | Display | ![]() |
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Full document | ![]() | 552.8 KB | Display | |
Data in XML | ![]() | 68.1 KB | Display | |
Data in CIF | ![]() | 103 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 35629MC ![]() 8h67C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Type I-MYXAN CRISPR-associated protein ... , 2 types, 2 molecules JA
#1: Protein | Mass: 63703.344 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: GenBank ID WP_071880939 / Source: (gene. exp.) ![]() ![]() Production host: ![]() ![]() References: UniProt: A0A068N831 |
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#2: Protein | Mass: 26592.424 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Production host: ![]() ![]() References: UniProt: A0A068N1Y0 |
-Protein , 2 types, 10 molecules BCDNOHIKLM
#3: Protein | Mass: 33789.074 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Production host: ![]() ![]() References: UniProt: A0A068N458 #8: Protein | Mass: 14538.500 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Production host: ![]() ![]() References: UniProt: A0A068N831 |
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-DNA chain , 3 types, 3 molecules EGP
#4: DNA chain | Mass: 4494.934 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: PAM proximal non target DNA / Source: (synth.) synthetic construct (others) |
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#6: DNA chain | Mass: 12725.236 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Target DNA / Source: (synth.) synthetic construct (others) |
#7: DNA chain | Mass: 3087.110 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: PAM distal non target DNA / Source: (synth.) synthetic construct (others) |
-RNA chain , 1 types, 1 molecules F
#5: RNA chain | Mass: 13975.212 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: CRISPR RNA / Source: (synth.) synthetic construct (others) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target presented as full R-loop formed state at 3.6 angstrom resolution Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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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: ![]() |
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) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92454 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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