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- In a separate column, assuming a linear buildup in time: chose now the first mixing time to get the initial cross-relaxation rate.
- Note, that this cross-relaxation rate is not normalized (it is not in [s-1]), but since we will do the referencing using a know distance, we do not have to normalize.
c: Calibrate the distances.
For the reference pair of protons. We have now also the reference sigma σRef.
Use the formula
rij = rRef * (σRef/σij)^(1/6). [Eq. 1] Vogeli 2014, Eq. 63b
to calculate the other distances between other atoms (in a separate column).
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- Try to explain what can be causing it.
- Check the median of the distance restraints placed into .upl
It should be around 4.2 Å for intramolecular distances, and 4.4 Å for the intermolecular distances. What do you get? - Discuss how robust the calculation of the protein-ligand structure is.
Introduction for task 2:
a:
The calibration of the distances from the measured NOE intensities, using a known, fixed distance, can be inaccurate for several reasons. In this exercise, we correct for some of them in two steps. Firts, the NOE intensities will be normalized using the diagonal intensities (their geometric mean) corresponding to the NOE crosspeak. This corrects for
- different lineshapes (if the peak height and not the peak volumes were taken)
- differences in the starting Z-magnetization (beginning of the NOE mixing time) due to incomplete relaxation between scans
- differences in the starting Z-magnetization due coherent transfer through other nuclei, such as in HSQC-NOESY.
b:
The This step above makes the crossrelaxation rates σiσi,j in correct mutual proportion. The actual values can be still inaccurate due to inaccurate reference distance, rRef in Eq. 1, or rather σRef being not correctly proportional to the rRef due to spin diffusion or other effects. It is therefore recommendable to correct the median of the measured distances such that it corresponds to the distances conserved among these organic molecules: around 4.2 Å for intramolecular distances and 4.4 Å for the intermolecular distances. When correcting the derived distances, we include a constant to multiply the distances in order to obtain the desired median.
c:
Task 2: Redo the normalization
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