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Instructions:
- download exercices.tgz, for example, to Documents, and untar (unpack them (in console, execute: cd Documents; tar xzvf exercises.tgz) them in some accessible folder.
- download the pythonScripts.tgz, if you wish to do the calculations yourself (many are time consuming).
- place the python directory, e.g., to the bin folder, and ensure that the bin/python is in the $PYTHONPATH:
- export PYTHONPATH=$PYTHONPATH:$HOME/bin/python
- lecture slides presentation.pdf
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- For viewing the molecular structures, we will use VMD.
module add vmd
- For NOESY simulations, we need a python environment.
module add anaconda3:2024.02
conda init
source .bashrc
conda activate base
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Introduction for
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Task 1:
In real experimental situation, we have never complete set of distances between each pair of protons. The NOE crosspeaks are detectable for distances up to around 5 Angstrom. From these, many signal would share the
same frequency in the spectrum, and thus, assignment between signal and atom (atom pair) can be done only within some group, or not at all. Furthermore, the experimentally-derived distances contain various sources of error.
Partly, it is due to random noise, but partly due to incompletely resolved relayed transfer and partly due to different (local) dynamics influencing the cross-relaxation rate.
Let's start anyway with the unrealistic situation, where we know all the distances within 5.5 A, accurately.
They are written Upper distance Limit files (here PxP.upl) file, which is used by CYANA.
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About CYANA
There is no closed-form formula to calculate the conformation (structure) from a set of distances. The setup starts with defining an energy penalty for every experimental distance not fulfilled by the molecular conformation. These are also called distance restraints. Starting from one chosen conformation, and trying to minimize the structure (using steepest descent or other local method) to fulfill the distances measured by NOE (or any other means) would fail: the structure would end-up in a local minimum. Instead we have to search for a global minimum. A commonly used algorithm for a global minimum is called simulated annealing, where the molecule is heated up such that high energy barriers (due to van der Waals clashes) can be surpassed. By a subsequent cooling, the imposed distance restraints will drive the molecule towards the conformation with minimal violation of the distance restraints. Many attempts will nevertheless end up in different local minima, and hence, only a subset of resulting conformers, the lowest-energy conformers will be likely to represent the global minimum.
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In this exercise, we gather together interatomic distances obtained before, separately from protein (030), for ligand (035), and add the protein-ligand distances. In this first attempt, we have exact distances (assume we are able to obtain them), with the exception of the protein-ligand distances, since here we do not account for the population concentration of the P, L and PL as discussed in (038), and hence the intermolecular calibration is incorrect, when using the known distance of protein atom pair. We will see the possible effect in this exercices
- In 000_cyana subdirectory, combine the ".upl" files obtained before like
cat PxP* PxL* LxL* > all.upl
- do the structure calculation (cyana CALC.cya) or check already the demo.ovw (cat demo.ovw)
- When viewing the complex using VMD, in Graphical representation, you can select the atoms of proteins by typing "protein" and the ligand as "not protein"
- Showing the protein structure as NewCartoon, we may miss a large portion, due to extra atoms - linker needed by CYANA to keep the ligand as a part of the same molecular graph with the protein
- Remove the linker (and other possible pseudoatoms) by
grep -v Q demo.pdb > demoClean.pdb
vmd demoClean.pdb
045 -
Here the populations (concentrations) are take into account correctly, so also the intermolecular distances are calibrated correctly.
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