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. 2022 Jan 25;298(3):101636. doi: 10.1016/j.jbc.2022.101636

Figure 1.

Figure 1

AcrIF13 targets the Cas5f-Cas8f tail of the Csy complex.A, MST assay of the binding between AcrIF13 and the Csy complex. AcrIF13 interacts with the Csy complex with a KD value equal to 1.51 ± 0.95 nM. B, untagged Acrs were mixed with the Csy complex containing 6xHis-tagged Cas7f. The samples were then affinity purified using Ni-NTA beads to remove unbound Acrs and proteins eluted from the beads and visualize through SDS-PAGE gel. In competitive binding experiments, one Acr was added after the first Acrs preincubated with Csy complex. An arrow means A replaced by B. C, native gel experiment for analyzing the interactions between Acrs (AcrIF2, AcrIF7, and AcrIF13) and the Cas5f-Cas8f heterodimer or the Csy complex. Acr protein (6 μM) (AcrIF2, AcrIF7, and AcrIF13) was incubated with different concentrations of Cas5f-Cas8f heterodimer of the Csy complex (3/6/12 μM). Lane 13 of the left panel was 12 μM Cas5f-Cas8f heterodimer alone. And then the mixture was visualized on 5% TBE native gels. Only AcrIF13 could not interact with Cas5f-Cas8f heterodimer. Acr, anti-CRISPR; Csy, CRISPR-Cas surveillance complex; MST, microscale thermophoresis.