EMD-23845

Single-particle
5.8 Å
EMD-23845 Deposition: 16/04/2021
Map released: 17/11/2021
Last modified: 29/05/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-23845

Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the Non-PAM side

EMD-23845

Single-particle
5.8 Å
EMD-23845 Deposition: 16/04/2021
Map released: 17/11/2021
Last modified: 29/05/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Geobacter sulfurreducens
Sample: Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the PAM side
Fitted models: 7mib (Avg. Q-score: 0.121)

Deposition Authors: Hu CY, Ke AK
Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.
Hu C , Almendros C, Nam KH , Costa AR , Vink JNA , Haagsma AC , Bagde SR , Brouns SJJ , Ke A
(2021) Nature , 598 , 515 - 520
PUBMED: 34588691
DOI: doi:10.1038/s41586-021-03951-z
ISSN: 1476-4687
ASTM: NATUAS
Abstract:
Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex2-4. 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)5,6 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.