Fig. 3: Analysis of the filament structure of the active NbaSPARDA complex.
From: Target DNA-induced filament formation and nuclease activation of SPARDA complex
a Overall structure of the active NbaSPARDA complex from two different views. b Topological structure of the filament in cartoon representation. Com.1, com.2, com.3, and com.4 represent the first, second, third, and fourth NbaSPARDA complex. PIWI–PIWI, MID–PIWI, and MID–MID interfaces were highlighted with arrows and labels. c Interaction details between the guide–target heteroduplex and NbaSPARDA complex. d Interaction details at the PIWI–PIWI interface. e Interaction details on the MID–PIWI and MID–MID interfaces. f Superposition of the structure of inactive NbaSPARDA complex onto one filament structure unit of the active complex. The key structural difference was shown alongside with zoomed-in view. g Plaque assay to evaluate the effect of key residue mutations on filament formation and E. coli’s defense against T5 bacteriophage invasion. DREN tetramer disruption mutant: N17A/E13A/R20A/R33A/Q29A/E45A/D31A; PIWI–PIWI disruption mutant: R287A/E253A/K324A/E360A/R285A/F256A. h FQ assay to determine the collateral nuclease activity of wild-type NbaSPARDA or mutants against ssDNA. FQ assay was performed in triplicate, and the error bars represent the standard deviations. Bridging loop extension: GGGGG linker inserted after aa 161; bridging loop deletion: aa 146–161 deletion; MID–MID disruption mutant: R244A/G140A/R142A/E97A; MID–PIWI disruption mutant: Q134A/R295A/R142A/D480A. i Plasmid interference assay to evaluate the effect of key residue mutations on plasmid transformation.
