Figure EV4. Effects of Mg²⁺ concentration on the DNA cleavage activity and the HNH location in the sgRNA/DNA‐Cas9 ternary complex.

• A
  Representative gel image of the DNA cleavage assay using the fluorescently labeled S355C–S867C construct. The sgRNA/DNA‐Cas9 ternary complex was incubated at room temperature (25°C) for 30 min. This condition is equivalent to that of the smFRET measurement, as we observed smFRET for approximately 30–40 min at room temperature.
• B
  Percentages of cleaved DNA against MgCl₂ concentration. The plot shows the results of four individual assays (black, blue, green, and red balls) for each MgCl₂ concentration. While the ternary complex with 0.5 or 1.0 mM MgCl₂ did not cleave the DNA, the complex with 2.0 and 5.0 mM MgCl₂ cleaved 39 ± 3% and 51 ± 6% (mean ± SEM, n = 4) of the DNA, respectively.
• C, D
  FRET efficiency histograms of fluctuating (C) and static (D) S355C–S867C molecules. The panels from top to bottom show data in the presence of 0.5, 1, and 5 mM MgCl₂. All of the assays were performed in the presence of 0.5 mM EDTA. The low, middle, and high FRET efficiencies corresponding the I, D*, and D positions of the HNH domain are indicated by black, green, and blue arrowheads, respectively (C). The numbers of observed molecules were 57, 72, and 40 for 0.5, 1, and 5 mM MgCl₂ conditions in (C), and 57, 55, and 70 for those in (D), respectively. The number of peaks in the histograms was estimated to be two for the histograms in (C) and one for the histograms in (D), using Silverman's test (threshold: P = 0.01). Thus, we fitted the histograms with two‐peak (C) or one‐peak (D) Gaussian distribution function (red curves). The peak values and widths of the higher FRET peaks were indicated on the histograms (median ± HWHM). The DNA cleavage activity (A, B) correlated well with the appearance of the highest FRET efficiency peak (C), indicating that the HNH domain in the D position adopts the cleavage‐competent state.