3. Pulse Scheme and Phase Cycle

4. Acquisition

Time requirement: 10.5 h Sample: 10 mg fully ¹³C- and ¹⁵N-labeled human ubiquitin in 600 yl 90% H,O/ 10% D,0, 50 mM phosphate (potassium salt), pH 5.8, 0.5 mM DSS.

Set and control the temperature to 300 K. A triple-resonance inverse probe-head with zgradient must be tuned to the sample on all three channels. ¹⁵N is assigned to the third hardware channel and ¹³C to the second. Note that the software uses different numbering; here protons are not in the first dimension, but in F3; the frequency dimension for CO is F; and for the ¹³C, region is F,. Record a 1D proton NMR spectrum without water suppression and adjust the offset on the water resonance. You have to set:

td3: 1024 data points in F; (¹H) td2: 32 data points in F2 (¹³C,) td: 32 data points in F; ('CO) sw3: 7 ppm sw2: 32 ppm swl: 20 ppm o1: on ¹H resonance of water [4.7 ppm] o2: middle of ¹³C NMR spectrum (C, region) [56 ppm]; provide a frequency list for ¹³C transmitter switching between the CO region [176 ppm] and the C, region. 03: middle of ¹⁵N NMR spectrum (amide region) [117 ppm] p1, p3, p6: 90° ¹H transmitter pulse [8 μs, 5 dB] p2, p4, p5, p7: 180° ¹H transmitter pulse [16 μs, 5 dB] p9, p14: 90° band-selective ¹³C decoupler pulse offset on Ca, q5 Gaussian cas- cade [400 μs, 1.2 dB] p8, p!7, p21: 180° band-selective ¹³C decoupler pulse offset on Ca, q3 Gaus- sian cascade [256 μs, | dB p1, p16: 90° band-selective ¹³C decoupler pulse offset on Ca, q5 Gaussian cascade, time-reversed shape to p9, p14 [400 μs, 1.5 dB] p18, p19, p23, p24: 180° band-selective ¹³C decoupler pulse offset on CO, q3 Gaussian cascade [256 μs, 1 dB] p20: 90° band-selective ¹³C decoupler pulse offset on CO, q5 Gaussian cas- cade [1.2 ms, 11 dB] pl0: 180° band-selective ¹³C decoupler pulse offset on C,, q3 Gaussian cas- cade, higher selectivity for refocusing of C, only [600 ys, 4.8 dB} pl2, p13: 180° band-selective ¹³C decoupler pulse on Cg, q3 Gaussian cascade