5MGA
Structure of the Cpf1 endonuclease R-loop complex after DNA cleavage
Summary for 5MGA
| Entry DOI | 10.2210/pdb5mga/pdb |
| Descriptor | CRISPR-associated endonuclease Cpf1, RNA (40-MER), DNA (26-MER), ... (6 entities in total) |
| Functional Keywords | fncpf1, cpf1, r-loop, crispr, hydrolase |
| Biological source | Francisella tularensis subsp. novicida U112 More |
| Total number of polymer chains | 4 |
| Total formula weight | 179330.73 |
| Authors | Montoya, G.,Stella, S. (deposition date: 2016-11-21, release date: 2017-06-21, Last modification date: 2024-05-08) |
| Primary citation | Stella, S.,Alcon, P.,Montoya, G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature, 546:559-563, 2017 Cited by PubMed Abstract: Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1. PubMed: 28562584DOI: 10.1038/nature22398 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (3 Å) |
Structure validation
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