Seitenhistorie

...

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

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

Part 1

Introduction for task 1:

...

• 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

  architectures, this same command can be used in all examples with protein and/or ligand)
  

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.

...

• 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 MHz
• What would be the consequence of "not so fast" exchange?
  1. for ligand structure calculation
  2. protein structure calculation
  3. protein-ligand distance extraction?
• experiment by modifying the parameters in parametersForKinMatrixDict.json
• run ./proteinLigandCalcNOESY.py triPepMod.pdb

Assuming 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.

039 (advanced intermezzo, no exchange!) - effect of pseudoatoms - chemically equivalent atoms

In this exercise, we will test the consequence of chemically equivalent atoms and hence the incomplete assignment of the NOESY spectrum, when deriving the cross-relaxation rates. This will be tested on a toy system with four atoms, of which, the middle two will be treated as 1) separate 2, pseudoatom containing both.

• ./pdbSimNoeFrontEnd.py triPep4H.pdb. (or cat pdbSimNoeFrontEnd.out)
• follow the three tasks to Compare. Answer namely,
• how,  in the NOESY spectrum, is the crosspeak between the first and last atom  affected by merging the middle ones?
• how is the extracted cross-relaxation rate (again between the first and last atom)  affected by this merging?

...

040 - structure of protein-ligand complex 

We 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), but we ignore the populations, so , with the exception of the protein-ligand distances, since here we do not account for the population of the P, L and PL as discussed in 038, and hence the intermolecular calibration is wrong.incorrect, when using the known distance of protein atom pair. We will see the possible effect in this exertices

• 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)
• Check the PxL.upl
• What are the effects when calculating the protein-ligand complex?
• 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 are take into account correctly, so also the intermolecular distances are calibrated correctly.

• What is the difference WRT 041 WRT 040 ?

solutions

...

Assembled by Dr. Jiří Mareš,shaped by discussion with Prof. Julien Orts, Florian Wolf and other members of the research group  (https://bionmr.univie.ac.at/people/)

...