Fig. 1.

Pulse sequences for 3D SOFAST HMQC-NOESY-HMQC with the following frequency labeling combinations: a) N(F₁)-N(F₂)H_N(F₃), b) C_M(F₁)-N(F₂)H_N(F₃), c) C_M(F₁)-C_M(F₂)H_M(F₃), and d) N(F₁)-C_M(F₂)H_M(F₃). The smaller and larger ¹H shaped pulses are 1.69 ms long 90° PC9_4_90 (Smith 2001) and 1.15 ms long 180° REBURP (Geen and Freeman 1991), respectively. The flip angle α (110° to 90°, depending on the value of d1 and the experiment type) of the shaped pulse after τ_mix should be optimized (see text). Depending on the sequence, the offsets of the ¹H shaped pulses are −3,230 Hz (at 0.9 ppm) for methyl type pulses and 3,230 Hz (at 8.5 ppm) for amides. The narrow bars represent 90° hard pulses. The shaped pulse on ¹³C channel represents a 500 µs long 180° smoothed CHIRP. (Hwang et al. 1997) The spectral centers of ¹⁵N, ¹³C, and ¹H dimensions are at 118.0, 16.5, and 4.7 ppm, respectively. The delays are: d1 = 0.2 sec, Δ1 = 1/(2×¹J_CH3) = 4.0 ms, Δ2 = 1/(2×¹J_NH) = 5.2 ms τ_mix = 0.3 sec. The phase cycling for X-XH-type are φ1 = (x, −x), φ2 = (x, −x, −x, x), φ_rec = (x, −x, −x, x) and for X-YH-type are φ1 = (x, −x), φ2 = (x, −x, −x, x), φ3 = 4(x), 4(−x), φ_rec = (x, −x, −x, x, −x, x, x, −x). Bruker decoupling scheme bi_garp_2pl is used. The quadrature detections in t1 and t2 dimensions are acquired via States-TPPI of φ1 and φ2, respectively. The durations and strengths of the gradients are G1 = (1 ms, 15 G/cm), G2 = (1 ms, 5 G/cm).