Modified STD3 pulse sequence Setup and Data Process

1D Saturation Transfer Difference (STD)  Experiment set up and process note:

  1. Login to vnmr1 account, and load the modified STD3 pulse sequence. (Just simply delete “hardloop macro”, and copy 40 guass shaped pulses instead, this modification can make “interleave method” work correctly.)
  2. Set satpwr=10dB, array satfrq=-0.2ppm,(-0.2+30)ppm, change the pw and tof to the calibrated values.

In order to process STD spectra, go to any non-exp5:

  1. ds(1) or df(1), and phase it, vs=vs*(100 or 10…), as needed.
  2. goto Process-> Add and Subtract 1D Data -> choose “Clear Buffer and Add Current Spectrum”
  3. ds(2) or df(2).
  4. goto Process-> Add and Subtract 1D Data -> choose “Add Second Spectrum into Buffer”
  5. click “-” button.
  6. click “->” button twice, and change it to blue color.
  7. now it shows the difference spectrum.

To save the fid files of the difference spectrum or the reference spectrum:

  1. goto any non-exp5, load the (STD3) spectra.
  2. type macro “clradd df(2) add df(1) sub jexp5″, and “enter”.
  3. now you can save the difference spectrum fid file in exp5.
  4. type macro “clradd df(2) add jexp5″, and “enter”.
  5. now you can save the reference spectrum fid file in exp5.

General considerations for STD exp.:

  1. Before STD exp., 1D proton spectrum of the protein receptor is needed, in order to make sure to put “satfrq” on the protein CH3 peaks’ region (in general).
  2. 1D proton spectrum of the ligand is needed, in order to avoid “satfrq” to be too close to the ligand’ peaks (at least 1.5ppm far away, to avoid artifact STD peaks).
  3. For the complex sample, the ligand concentration should be ~50 (10 to 100) folds more than the protein’s for high ligand access (increasing the possibility of saturation transferring from one protein molecule to more ligand molecules), and increase the sensitivity.
  4. The size of the protein should be big enough to meet the spin diffusion (or slow tumbling) limit (ω2τc2 » 1).
  5. If the dissociation constant KD of ligand-receptor system is from 10-8 to 10-3 mol/L, the STD is working well; for the very strong binding, STD is not working well because of non-effective saturation transferring.
  6. Saturation time should be long enough, 2 sec here (up to 3 sec).

updated on Aug.04, 2014
last updated on Jan.02, 2014

Comments are closed.