Seitenhistorie

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The NOESY spectrum of the ligand  (the intramolecular NOEs) would be formed as a population  average of the bound and free form. In the free form, cross-relaxation rate is commonly negative, whereas in the bound form, it is positive, the same as the protein. Moreover, it is commonly much larger (absolute value).

035

• For the case of 1:10, 1:1 and 1:200 of protein:ligand fractions, in 035, 035...LessOfLigand and 035...MoreOfLigand.
• Check the simulated NOESY spectra (!note that the axes are not chemical shifts but simply atomic indices!)
  • see the file: NOESYZoomLigand.pdf, the right lower corner is the section corresponding to the ligand.
• How would you determine structure of a ligand in the bound state?

038 

The exercise above assumed that the exchange rate between free and bound states is large. Here we will look at a toy system of 3 protons representing a protein, and 2 protons representing a ligand, varying the exchange rate and it's effect on the simulated NOESY spectrum. 

• Print the content of the file on the screen 

  • cat NOESYwhenDifferentExchangeRates.txt

  • (or you can extract these using: grep -A 5 "NOESY intensities s" outputKex5e*t10)

•  Look at the last column, last two rows, corresponding to the ligand NOE signals and note their intensity for different exchange frequencies - between 5 Hz and 5 MHzWe check the effect of the exchange rate on the resulting cross-relaxation rates.
  See e.g. like: tail -n 15 outputKex5e*
• What would be the consequence of "not so fast" exchange?
  1. for ligand structure calculation
  2. protein structure calculatioin
  3. protein-ligand distance extraction?
• experiment by modifying the parameters in parametersForKinMatrixDict.json
• run ./proteinLigandCalcNOESY.py triPepMod.pdb
• experiment with settings in parameters*.json files.

What is even more important is the population weighting of the intermolecular cross-rates (hence also NOEs)
The "Re weighted" matrix contains includes the correction, whereas the "weight averaged over P, L, PL" does notAssuming the fast exchange, the derive the correct intermolecular distances between the protein and ligand, the extracted cross-relaxation rates have to be scaled by the population of the components (P, L, PL). The formulas are in presentation, whereas the code is used in the end if the python script. You can check it if there is time left.

041 

We have exact distances (assume we are able to obtain them), but we ignore the populations, so the intermolecular calibration is wrong.

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