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.

Instructions:

• download exercicesexercises.tgz, for example, to Documents, and unpack them (in console/terminal, execute: cd Documents; tar xzvf exercises.tgz).
• 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. In steps: create by mkdir bin, then, if the scripts were in the Downloads folder, use mv  Downloads/python.tgz bin; cd bin; tar xzvf python.tgz, and ensure that the bin/python is in the $PYTHONPATH:
  • in terminal, execute: export PYTHONPATH=$PYTHONPATH:$HOME/bin/python
• lecture slides presentation.pdf

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• For viewing the molecular structures, we will use VMD.  In console/terminal execute:
  

  • module

    add

    vmd

• For NOESY simulations, we need a python environment. Execute following commands ()
  

  • module

    add

    anaconda3:2024.02

  • conda

  • init

  • source

  • .bashrc

  • conda

  • activate

  • base

Introduction for 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.

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• 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 (cat demo.ovw)
  See the target functions, its variation.
  See the RMSD - root mean square displacement.
• To view the structure, use

  vmd

  demo.pdb #the same name will be produced in all trials of this tutorial
  

  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 using a ready protein-ligand structure (final.pdb), in the 030 folder,

  run ./proteinLigandCalcNOESY.py final.pdb (can take 20 min on some architectures, this same command can be used in all examples with protein and/or ligand) obviously doing different tasks.
  

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 Angstrom for the strongest and 5.5 to the weakest.

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• Check some of the simulated buildups, the buildupsLinearized in the supplied folders.
• Without further effort, what are the chances to get accurate distances from these?
• Check how many distance restraints we have: wc  wc -l PxPapp.upl
• Start the structure calculation, or see the ready demo.pdb and demo.ovw file.

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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. 

• Look at the coordinates (3 and 2 atoms extracted from a protein structure) cat triPepMod.pdb
• 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 (open in some text editor, such as )
• run ./proteinLigandCalcNOESY.py triPepMod.pdb

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• 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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