4. Acquisition

Time requirement: 39 h Sample: 10 mg fully ¹³C- and ¹⁵N-labeled human ubiquitin in 600 p1 90% H2O/ 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 z-gradient 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 in F3; the frequency dimension for ¹⁵N is F2 and that for 3C is F,. Record a 1D proton NMR spectrum and adjust the offset on the water signal. You have to set:

td3: 2k data points in F; (¹H) td2: 64 data points in F, (N) td1: 64 data points in F, (¹³C) sw3: 12 ppm sw2: 40 ppm sw1: 67 ppm o1: on water resonance [4.7 ppm] o2: middle of ¹³C NMR spectrum (Cap region) [38.5 ppm] 03: middle of ¹⁵N NMR spectrum (amide region) [117 ppm] p1, p3, p5, p7, p9, p11, p13, p15, p17: 90° ¹H transmitter pulse [9 μs, 5 dB] p2, p4, p6, p10, p12, p14, p16, p18: 180° ¹H transmitter pulse [18 μs, 5 dB] p8: 90° selective rectangular-shaped ¹H pulse on water signal [2 ms, 53 dB] p20, p22: 90° ¹³C decoupler pulse [21 μs, 0 dB] p21: 180° ¹³C decoupler pulse [42 μs, 0 dB] p19, p23, p26: adiabatic 180° ¹³C decoupler pulse, offset 75 ppm, (crp 60, 0.5, 20.1) [500 μs, 1.7 dB] p24, p25: selective 180° ¹³C decoupler pulse, offset 170 ppm, q3 Gaussian cascade [256 μs, 0.5 dB] p29, p31, p33: 90° ¹⁵N decoupler pulse [30 pis, 2 dB] p27, p28, p30, p32, p34: 180° ¹⁵N decoupler pulse [60 pss, 2 dB] dl:2s d2: 1/[4(C,H)] = 1.47 ms, calculated from ¹³C,H) = 145 Hz, corrected for length of p19/2 d3: NOE mixing time, 70 ms – effective gradient duration d4: 1/[4J(N,H)] = 2.3 ms, calculated from 'J(N,H) = 90 Hz with compromise for relaxation dS: effective gradient duration = 1.05 ms d6: 1/[8/(N,H)] = 1.38 ms for all multiplicities g|l–g6: sinusoidal-shaped field gradients of 1 ms length, ca. 0.1 T/m strength,

with gradient loop counters, ring-down delays (50 μs), lock blanking and gradient coil blanking switches according to actual instrumentation used, g5 with sign alternation according to echo/anti-echo.