Figure 4.

ZF-TEDOR spectra collected at 600 MHz with DNP are contoured at two mixing times in (a), and displayed for the G34 cross-peak as a function of mixing time in (b). At 3.84 ms mixing (a, left), one bond cross-peaks are detected due to the natural abundance of ~1% ¹³C Rmt drug, and ~0.36% ¹⁵N protein. By 11.52 ms (a, right), these natural abundance cross-peaks have decayed and only the cross-peak of interest remains. In (b), the G34 cross-peak intensity is shown as a function of mixing time (points ± the spectrum rms noise level), with the best-fit simulation as described in the text (smooth curve). The fit resulted in a distance of 4.0 Å ± 0.2 Å. The G34 N-Cα cross-peak was simultaneously fit (b, right) to reduce the degrees of freedom in the fit. A low natural abundance of ¹⁵N (~0.36%) accounts for the reduced signal of this curve. The spectra were recorded in ~4 days at a spinning frequency of 12.5 kHz and a temperature of ~100 K. The DNP enhancement factor was 2.5.