Fig. 4: Structural basis for slow Cas9 dissociation.
From: Visualization of a multi-turnover Cas9 after product release

a Cryo-EM map of the dissociated state colored by domain. b Zoom-in of the pre-ordered sgRNA seed region. c Zoom-in view of the PAM contacts maintained even following PAM-distal product release. d Schematic of the nucleic acid in this complex. sgRNA colored in red, target strand in green, and non-target strand in pink. PAM site indicated with gray box. e Modevector arrows showing the domain movements from the checkpoint to dissociated states. f Monitoring PAM-distal and PAM-proximal product release following cleavage. Cas9 programmed with a 20-, 17-, 15-nt, or 2 mm sgRNA was incubated with target DNA labeled where the PAM-distal end was labeled with FAM and the PAM-proximal end labeled with Cy5. One sample containing 5X molar excess Cas9 20-nt RNP was included and treated with Proteinase K (+PK) as a control. Product release was analyzed at 2 h following reaction initiation on Native 4-20% polyacrylamide gels. Samples were quantified using ImageJ and plotted as a ratio of released product to bound product and represented as a percentage. Data represents the mean ± s.d. with n = 3. Statistical significance was determined using a one-way ANOVA with the Dunnett correction for multiple comparisons. ****p = 2.74e−8. Source data are provided as a Source Data file.