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. 2022 Oct 16;50(19):11344–11358. doi: 10.1093/nar/gkac880

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — AcrIF24 directly binds type I-F Cascade via Cas7f1. (A) Schematic showing the composition and interference process by the type I-F Cascade complex. The crRNA processed and bound by Cas6f acts as a scaffold for type I-F Cascade assembly. (B) Interaction analysis between AcrIF24 and Cascade by SEC. SEC profiles produced by AcrIF24 (blue line), Cascade (red line), and the mixture of AcrIF24 and Cascade (black line) are shown. (C) SDS-PAGE gels produced by loading one of the main fractions from Cascade sample with (+AcrIF24) or without (–AcrIF24) providing AcrIF24. The position of each Cascade subunit on the gel is indicated. AcrIF24 co-migrated with Cascade is indicated by a red arrow. (D) Position of mutated residues marked on the dimeric structure of AcrIF24. Mutated residues G22Y (representative wing domain disruption mutant), D105K and W110K (representative head domain disruption mutants), and G189K (representative body domain disruption mutant) were indicated by red color. (E) Interaction analysis of Cascade with wildtype AcrIF24 and with various mutants of AcrIF24 by SEC followed by SDS-PAGE. SDS-PAGE gels produced by loading one of the main fractions from Cascade sample with various mutants of AcrIF24. The position of each Cascade subunit and AcrIF24 on the gel is indicated. The reduced co-purification of AcrIF24 (W110K) with Cascade is denoted on the gel by red dot-box. The tentative position of the ΔHead mutant that did not co-migrate with Cascade is also denoted in the gel by red dot-box. (F) Bar-chart showing the quantified intensity of the co-eluted AcrIF24 and various mutants on SDS-PAGE provided in (E). Data are presented as the mean ± standard deviation of three independent experiments. (G–I) Interaction analysis between AcrIF24 and each Cascade subunit by SEC. SEC profiles produced by AcrIF24 (blue line), each Cascade subunit (red line) of Cas5f1 (G), Cas6f (H) and Cas7f1 (I), and the mixture of AcrIF24 and each Cascade subunit (black line) are shown. SDS-PAGE gel produced by the mixture of AcrIF24 and each Cascade subunit is provided under the SEC profile. Loaded fractions for SDS-PAGE were indicated by a black bar.