Fig. 3.

a–c Schematic presentation of magnetization transfer pathway during the 4D iHACANCO d, 4D HACACON e and 4D (HACA)CONCAHA f experiments. Red arrows indicate direct transfer pathway from ¹H^α(i) to ¹⁵N(i) or ¹⁵N(i + 1) whereas green arrows indicate a nested C^αC′_ZN_Z →N_ZC^α transfer in d) known as the intraresidual filter (Permi 2002; Brutscher 2002) or highly selective C^αC′_ZN_Z →C^α transfer in e). Arrows indicate out-and-back type magnetization transfer, whereas one-way arrows represent coherence transfer route which is unidirectional. One-letter codes above the arrows indicate time points in the pulse sequence. d Intraresidual iHACANCO experiment to correlate ¹H^α(i), ¹³C^α(i), ¹³C′(i) and ¹⁵N(i) chemical shifts, e the HACACON experiment, which correlates chemical shifts of ¹H^α(i), ¹³C^α(i), ¹³C′(i) and ¹⁵N(i + 1) resonances. f The (HACA)CONCAHA experiment for correlating ¹H^α(i), ¹³C^α(i), ¹³C′(i) and ¹⁵N(i + 1) resonances. Narrow and wide filled bars on ¹H and ¹⁵N channels correspond to rectangular 90° and 180° pulses, respectively, applied with phase x unless otherwise stated. All ¹³C pulses are band-selective shaped pulses, denoted by filled narrow bars (90°) and filled and unfilled half ellipsoids (180°). Unfilled bars are applied on-resonance. The ¹H, ¹⁵N, ¹³C′, and ¹³C^α carrier positions are 4.7 (water), 118 (center of ¹⁵N spectral region), 174 ppm (center of ¹³C′ spectral region), and 56 ppm (center of ¹³C^α spectral region). The ¹³C carrier is set initially to the middle of ¹³C′ region (174 ppm), shifted to ¹³C^α region (56 ppm) prior to 90° ¹⁵N pulse ϕ₁ in scheme d). In scheme e) and f), the carrier is initially at 56 ppm and shifted to 174 ppm prior to 90° ¹³C pulse ϕ₂, and shifted back to 56 ppm before 90° ¹³C pulse ϕ₄. The first band-selective 180° ¹³C pulse, refocusing ¹³C^α magnetization (56 ppm, denoted with an asterisk) had duration of 788 μs at 800 MHz. Other band-selective 90° and 180° pulses for ¹³C^α (56 ppm) and ¹³C′ (174 ppm) were applied with durations of 240.0 μs and 192.0 μs at 800 MHz, respectively. Band-selective 90° and 180° pulses for ¹³C′/¹³C^α have the shape of Q5 and Q3 (Emsley and Bodenhausen 1992) and duration of 240.0 μs and 192.0 μs at 800 MHz, respectively. The adiabatic 180° Chirp broadband inversion pulse for inverting ¹³C^α and ¹³C′ magnetization in the middle of t₁ period had duration of 500 μs at 800 MHz (Böhlen and Bodenhausen 1993). The Waltz-65 sequence (Zhou et al. 2007) with strength of 4.17 kHz was employed to decouple ¹H spins. The GARP (Shaka et al. 1985, 1987) with field strength of 4.55 kHz was used to decouple ¹³C during acquisition. Delay durations: τ = 1/(4J_HC) ~ 1.7 ms; τ₂ = 3.4 ms (optimized for non-glycine residues) or 2.2–2.6 ms (for observing both glycine and non-glycine residues); ε = duration of G_H + field recovery ~ 0.4 ms; 2T_C = 1/(2J_Cα_C′) ~ 9.5 ms; T_CA = 1/(6J_Cα_C′) ~ 3.3 ms; T_A = 1/(4J_C′N) ~ 16.6 ms; T_C′ = T_C + T_CC; T_CC = 1/(J_Cα_Cβ)–1/(4J_C′N)–1/(2J_Cα_C′) ~ 0–2.5 ms; T_NC ~ 14 ms; T_CN ~ 14 ms; T_N ~ 14 ms. Maximum t₁, t₂ and t₃ are restrained in scheme d, t_2,max < 2.0*T_C′, t_3,max < 2.0*T_CN, in scheme e, t_2,max < 4.0*T_A, t_3,max < 2.0*T_CA, in scheme f, t_1,max < 2.0*T_A, t_2,max < 2.0*T_NC, t_3,max < 2.0*T_CN. Frequency discrimination in ¹⁵ N and ¹³C′ dimensions is obtained using the States-TPPI protocol (Marion et al. 1989) applied to ϕ₁ and ϕ₂, respectively, whereas the quadrature detection in ¹³C^α dimension is obtained using the sensitivity-enhanced gradient selection (Kay et al. 1992; Schleucher et al. 1994). The echo and antiecho signals in ¹³C^α dimension are collected separately by inverting the sign of the G_C gradient pulse together with the inversion of ψ, respectively. Phase cycling: ϕ₁ = x, − x; ϕ₂ = 2(x), 2(− x); ϕ₃ = 4(x), 4(− x); ϕ₄ = x; ψ = x; rec. = x, 2(− x), x, − x, 2(x), − x. Selective 180° pulse for ¹³C^α in the middle of delay 2T_A induces a Bloch-Siegert shift to ¹³C′ magnetization, a careful adjustment of phase (bsp) of the last ¹³C′ 90° (phase y) pulse is necessary in scheme d). Gradient strengths and durations: G_C = 13 k G/cm (1.6 ms), G_H = 13 k G/cm (0.4 ms). The pulse sequences code and parameter file for Bruker Avance system are available from authors upon request