Seitenhistorie

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1. learn how to calibrate NOE data to obtain distance restraints,
2. derive a protein structure with the software CYANA,
3. try how the exchange of the ligand between free and bound state influence the NOESY spectra,
4. derive the structure of protein-ligand complex,
5. try how the incomplete assignment due to chemically identical atoms affect the NOESY simulation.

We will use the software CYANA. At wolf.ncbr.muni.cz

• 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

Instructions:

• download exercices.tgz to and untar (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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Remark: CYANA is a proprietary software. For any installation problem, contact Peter Güntert, the author of CYANA.

Task 1 (030)

• In 000_...cyana subfolder, look into the configuration file, CALC.cya, using a text editor/browser.
  The set of structures will be written in demo.pdb
• Start the calculation by

  cyana CALC.cya

• After the calculation is ready, look at the .owv file.
  See the target functions, its variation.
  See the RMSD - root mean square displacement.
• To view the structure, use

  vmd demo.pdb

  In VMD, the default view shows the interatomic bonds as lines.
• Go to Graphics->Representations
  and change the Drawing Method to CPK.
  To see the common representation for proteins, choose NewCartoon as the Drawing method.
  See how alpha-helices, beta-sheets and loops are identified.
• To simulate the NOESY spectrum, in the 030 folder,

  run ./proteinLigandCalcNOESY.py final.pdb (can take 20 min on some architectures)

Introduction to Task 2

It used to be common to only simply classify the experimental NOE intensities to strong, medium and weak, and assign corresponding distance ranges of around 2 Angsrom for the strongest and 5.5 to the weakest.

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Here we probe three effortless options to obtain a better set of distances. The first comes from an idea, that short distances are much less likely to be affected by relayed transfer (spin diffusion), so we try to keep only those, within 2.5 Angstrom. Furthermore, to be safer, the approximate distances are multiplied by 1.5

• Check how many distances are left.
• Visualize the structure in VMD, intead instead of using only one Drawing Method, press Create Rep in Graphical representation. Keep one as Lines and other as NewCartoon.
• (Doublecklick on one of the copy would hide its visibility.)
• What can be problem when using only this short distances?
• Is the result satisfactory?

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034

In the next simple attempt, we multiply all the distances by a constant factor (1.75) and use again those within 5.5A.

• Check how many are left, what is the statistics of the structure calculation. And
• visualize the structure.
• Switch again to NewCartoon drawing method. What happens?

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Next, we can think that the number of (inaccurate) constraints is simply too large.   Commonly we would expect up to
around 10 constraints per aminoacid residue.  In the previous example, it was still around 12800/97 > 100 restraints
per residue. Here we try to use, only every 10th restraints.

• See Check how many are left, what is the statistics of the structure calculation overview. And
• visualize Visualize the structure.
• Switch again to NewCartoon drawing method. What happens?
• What do you think, were these attempts good enough?

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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).

• Check the simulated NOESY spectra (!note that the axes are not chemical shifts but simply atomic indices!)
• For the case of 1:10, 1:1 and 1:200 of protein:ligand fractions, in 035, 036 and 037.035...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
• What more is not realistic about this simulation?

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