4. Acquisition

Time requirement: 30 h

Sample: 10 mg fully ¹³C- and ¹⁵N-labeled human ubiquitin in 600 μl 90% H₂O/10% D₂O, 50 mM phosphate (potassium salt), pH 5.8, 0.5 mM DSS.

A triple-resonance inverse probe-head with z-gradient must be tuned to the sample on all three channels. Usually 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; ¹⁵N is in F₂ and ¹³C in F₁. Set and control the temperature to 300 K. Record a 1D proton NMR spectrum without water suppression and determine the water frequency. You have to set:

td3: 2048 data points in F₃ (¹H)
td2: 32 data points in F₂ (¹⁵N)
td1: 64 data points in F₁ (¹³C)
sw3: 12 ppm
sw2: 40 ppm
sw1: 30 ppm
o1: on resonance of water signal [4.7 ppm]
o2: middle of ¹³C NMR spectrum (C_α region) [56 ppm]
o3: middle of ¹⁵N NMR spectrum (amide region) [117 ppm]
p1, p3, p9, p11, p13: 90° ¹H transmitter pulse [8 μs, 5 dB]
p2, p7, p8, p10, p12, p14: 180° ¹H transmitter pulse [16 μs, 5 dB]
p4: 90° selective rectangular-shaped ¹H transmitter pulse, offset on water frequency [2 ms, 53 dB]
p5, p6: 90° ¹H transmitter pulse at power level of DIPSI-2 spin-lock [70 μs, 23.5 dB]
p16, p18, p20, p22, p24: 90° ¹⁵N decoupler pulse [30 μs, 2 dB]
p15, p17, p19, p21, p23, p25: 180° ¹⁵N decoupler pulse [60 μs, 2 dB]
p27: 90° band selective ¹³C decoupler pulse, offset on C_α, q5 Gaussian cascade [400 μs, 1 dB]
p29: 90° band-selective ¹³C decoupler pulse, offset on C_α, q5 Gaussian cascade, time-reversed shape to p27 [400 μs, 1 dB]
p26, p28, p30: 180° band-selective ¹³C decoupler pulse, offset on C_α, q3 Gaussian cascade [256 μs, 0 dB]
p31, p32, p33: 180° band-selective ¹³C decoupler pulse, offset on CO (176 ppm), q3 Gaussian cascade [256 μs, 0 dB]
d1: 2 s
d2: 1/[4J(N,H)] = 2.3 ms, calculated from ¹J(N,H) with compromise for relaxation
d3: 1/[2J(N,H)] = 5.5 ms, calculated from ¹J(N,H) = 90 Hz
d4: 1/[4J(N,C_α)] − 1/[2J(N,H)] = 6.5 ms
d5: 1/[4J(N,C_α)] = 12 ms, calculated from ¹J(N,C_α) with compromise for relaxation