How to setup STD Expt.

If using 90%H2O/10%D2O(or DMSO-d6),  prepare three samples:

  1. only the ligand.
  2. only the protein receptor (MW: >20 kDa, the size of the receptor should be big enough to meet the spin diffusion or slow tumbling limit: ω2τc2 » 1).
  3. the mixture of the ligand and the receptor ([L] should be 50 (10 to 100) folds more than [R] for high ligand access, namely, increasing the possibility of saturation transferring from one protein molecule to more ligand molecules, and increase the sensitivity):

NMR operation:

  1. Run ZGPR first, find p1 by pulsecal, o1 by gs, and find proper sw.
  2. Run 1D ZGESGP for the receptor sample, make sure to put “satfrq” on the protein CH3 region, in general.
  3. Run 1D ZGESGP for the ligand sample, and avoid “satfrq” to be too close to any ligand’s peaks (at least 1.5ppm far away, to avoid artifact STD peaks).
  4. Load STDDIFFESGP.3; for the shape pulse sp9, select Gaus1.1000 and set 50dB, (40 to 60dB), shape pulse length p42=50ms; the saturation time d20 should be long enough, d20=2s here (up to 3s).
  5. Type fq2list, edit the file 3 lines like: bf ppm; 0.78p; 30.78p, and saved as STD_2satfrqs filename, retype fq2list again and select this file.  d1=2s, rg=203 since using excitation sculpting.

STD difference spectrum processing:

  1. Drag in raw data, efp, input 1 and PROCNO as 101; apk, .ph, save and exit.
  2. Drag in raw data again, efp, input 2 and PROCNO as 102; apk, .ph, save and exit.
  3. Type .md, drag in both of 101 and 102, click ∆, and save as 103, which is the STD difference spectrum.


If the dissociation constant KD of ligand-receptor system is from 10-8 to 10-3 mol/L, the STD is working well; for much stronger binding (KD ~ nM), STD is not working well because of non-effective saturation transferring.

Hongwei, Edited on 6/7/2023