7MI4
Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Summary for 7MI4
| Entry DOI | 10.2210/pdb7mi4/pdb |
| EMDB information | 23839 |
| Descriptor | CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion, CRISPR-associated endoribonuclease Cas2, DNA (35-MER), ... (5 entities in total) |
| Functional Keywords | crispr/cas, cas4, pam recognition, hydrolase-dna complex, hydrolase/dna |
| Biological source | Geobacter sulfurreducens More |
| Total number of polymer chains | 8 |
| Total formula weight | 295456.95 |
| Authors | |
| Primary citation | Hu, C.,Almendros, C.,Nam, K.H.,Costa, A.R.,Vink, J.N.A.,Haagsma, A.C.,Bagde, S.R.,Brouns, S.J.J.,Ke, A. Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas. Nature, 598:515-520, 2021 Cited by PubMed Abstract: Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. PubMed: 34588691DOI: 10.1038/s41586-021-03951-z PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (3.2 Å) |
Structure validation
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