Fig. 4.
Rapid conformational fluctuations coupled with TS cleavage activity. (A, Left) Schematic for Cas9LD750-dRuvC, which cleaves TS. (A, Right) Representative FRET trace from the DNA landing assay using Cas9LD750-dRuvC, acquired at 10 min after DNA binding to Cas9. The FRET efficiency trajectory (blue) was further idealized (red) using vbFRET (32). (B) Transition density plot from the DNA landing assay using Cas9LD750-dRuvC. The FRET transitions were identified from 100 single-molecule FRET traces acquired at 10 min after DNA binding using vbFRET. Transition rates between states are indicated in Fig. 4B, which were extracted from survival probability plots in SI Appendix, Fig. S12A. (C) FRET histogram from the DNA landing assay Cas9LD750-dRuvC, acquired at 10 min after DNA binding to Cas9. Green or red curve represents a fit to a single Gaussian peak. Blue curve represents a fit to double Gaussian peaks. (D, Left) Schematic for Cas9LD750, which cleaves both DNA strands. (D, Right) Representative FRET trace from the DNA landing assay using Cas9LD750, acquired at 10 min after DNA binding to Cas9. The FRET efficiency trajectory (blue) was further idealized (red) using vbFRET. (E) Transition density plot from the DNA landing assay using Cas9LD750. The FRET transitions were identified from 157 single-molecule FRET traces acquired at 10 min after DNA binding using vbFRET. Transition rates between states are indicated in Fig. 4E, which were extracted from survival probability plots in SI Appendix, Fig. S12B. (F) FRET histogram from the DNA landing assay Cas9LD750, acquired at 10 min after DNA binding to Cas9. Green or red curve represents a fit to a single Gaussian peak. Blue curve represents a fit to double Gaussian peaks. (G) Model for Cas9-mediated DNA cleavage. Cas9 is gray colored, except the HNH domain is yellow colored. Guide RNA, TS, and NTS are orange, blue, and light purple.