Fig. 1.

Description of the HACANCOi experiment. a Schematic presentation of magnetization transfer pathway during the proposed 4D HACANCOi experiment and 4D iHACANCO scheme (Mäntylahti et al. 2010; Tossavainen et al. 2020). Black arrows indicate the so-called out-and-back transfer pathway from ¹H^α(i) to ¹³C^α(i) and further to ¹³C′(i) and ¹⁵N(i)/¹⁵N(i + 1). b 4D HACANCOi experiment to correlate ¹H^α(i), ¹³C^α(i), ¹³C′(i) and ¹⁵N(i)/¹⁵N(i + 1) chemical shifts. Inset b′ 3D HA(CA)NCOi experiment to correlate ¹H^α(i), ¹³C′(i) and ¹⁵N(i)/¹⁵N(i + 1) chemical shifts. 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°). Pulses denoted with unfilled bars are applied on-resonance. The ¹H, ¹⁵N, ¹³C′, and ¹³C^α carrier positions are 4.7 (water), 121 (center of ¹⁵N spectral region), 174 ppm (center of ¹³C′ spectral region), and 54 ppm (center of ¹³C^α spectral region). The ¹³C carrier is initially set to the middle of ¹³C′ region (174 ppm), and shifted to ¹³C^α region (54 ppm) prior to 90° ¹⁵N pulse ϕ₃. All band-selective 90° and 180° pulses for ¹³C^α (54 ppm) and ¹³C′ (174 ppm) 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, denoted with striped half ellipsoid in both ¹³C channels, for inverting ¹³C^α and ¹³C′ magnetization in the middle of t₂ 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 ~ 1.2 ms; 2T_C = 1/(2J_CαC’) ~ 9.5 ms; 2T_CAN ~ 28 ms. Maximum t₃ is restrained t_3,max < 2.0*(T_CAN–τ₂). Frequency discrimination in ¹³C′ and ¹⁵N 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(y), 4(− y); ϕ₄ = y; ψ = x; rec. = x, 2(− x), x, − x, 2(x), − x. Gradient strengths (% of max G/cm) and durations (ms): G₁ = 17%, 0.234 ms; G₂ = 40%, 1.0 ms; G₃ = 60%, 1.0 ms; G₄ = 25%, 1.0 ms; G₅ = 35.7%, 0.234 ms; G₆ = 7.1%, 0.234 ms; G_C = 80.0%, 1.0 ms; G_H = 20.1% 1.0 ms. The pulse sequences’ code and parameter file for Bruker Avance system are available from authors upon request