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Lecture slides can be downloaded here: presentation.pdf
At wolf.ncbr.muni.cz, paste these commands (from "module add vmd" to "cd exercises") to the terminal/console and press enter (for other machine, follow the instructions at the end of the page).
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Get a copy of the python scripts and of the exercises:
- download exercises.tgz, for example, to your home (~/), and unpack them (in console/terminal, execute: tar xzvf exercises.tgz).
- download the pythonScripts.tgz, if you wish to do the calculations yourself (many are somewhat 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
- be sure to have also these python libraries installed:
- scipy, numpy, networkx, sympy, matplotlib, ipython
- (at the moment, one library of the pythonScripts.tgz is only as a binary, so unfortunately, the exercises may fully work only with python3.11 and 3.12, please contact me if you encounter problems and if you would like to have the scripts working in your environment)
Installing an additional software:
- for visualization of molecules, install vmd https://www.ks.uiuc.edu/Research/vmd/
- for structure calculation using NOESY-derived restraints, install cyana http://www.cyana.org/wiki/Main_Page
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Introduction for Task 1:
In a 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 at the configuration file, CALC.cya..
- ( execute in terminal/console: cd 030_proteinGetExactDistancesProtein/000_cyana )
- cat CALC.cya
- Start the calculation by
cyana CALC.cya
- The set of structures will be written in demo.pdb
- 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 but always starting with assembling the relaxation matrix using the given ".pdb" coordinates, and proceeding with simulation of one or several NOESY spectra.
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 Angstrom for the strongest and 5.5 to the weakest. Here we try to get the actual distances.
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Here we use a technique, which would be a starting point for more accurate methods. It uses spectra recorded for different mixing times. As the NOESY spectrum of a protein can take days to record, recording series of them is a large investment. In that the NOE crosspeak volume (intensity) is divided by a geometric mean of the corresponding diagonal volumes. The slope is a better approximation of the crossrelaxation rate than if this "linearization" or "normalization" was not done. In the case of two isolated nuclei, such buildup curve is approximately linear over longer mixing times. We again start with simulation of the NOESY spectrum instead of having an experimental one.
- Check some of the simulated buildups, the buildupsLinearized in the supplied folders.
- cd ~/exercises/031_getApproximateDistancesProtein/buildupsLinarized
- open buidupsLin.pdf
- Without further effort, what are the chances to get accurate distances from these?
- Check how many distance restraints we have:
- cd ../000_cyana
- wc -l PxPapp.upl
- Start the structure calculation (cyana CALC.cya), or see the ready demo.pdb and demo.ovw file.
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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).
035 - structure of a ligand
- For the case of 1:10, 1:1 and 1:200 of protein:ligand fractions, in 035, 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, the right lower corner is the section corresponding to the ligand.
- cd ~/exercicesexercises/035_genLigandDistancesExact
- open NOESYZoomLigand.pdf
- what is the sign and intensity of the NOESY crosspeaks of the ligand - indices around 120 to the end? (compared the part of the protein)
- Check now with lower concentration of the ligand (1:1 ration with protein)
- cd ~/exercicesexercises/035_genLigandDistancesExactLessOfLigand
- open NOESYZoomLigand.pdf
- what is not the sign and intensity of the NOESY crosspeaks of the ligand?
- Check with a high concentration of the protein:
- cd ~/ exercices
- exercises/035_genLigandDistancesExactMoreOfLigand
- open NOESYZoomLigand.pdf
- the same question
- Based on these simulated spectra, how would you determine structure of a ligand in the bound state?
038 - effect of the exchange on NOESY spectra (a toy system)
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)
- cd ~/exercises/038_toySystemWeightIntermolecularNoes
- 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?
- for ligand structure calculation
- protein structure calculation
- protein-ligand distance extraction?
- experiment by modifying the parameters in parametersForKinMatrixDict.json (open in some text editor, such as vim)
- run ./proteinLigandCalcNOESY.py triPepMod.pdb
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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(printed by the script) to compare. Answer namely,
- how, in the (simulated) 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?
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- See the time-evolution of the cross-peak (first and last atom) as plotted in "relayedThroughHHorQ.pdf". What does it mean?
- open relayedThroughHHorQ.pdf
- In the script, change the "specIndex" from 10 to 50; now the NOESY will be simulated for tauMixing=2.5s
- rerun the script. Notice the changes in the output.
- see also the new "relayedThroughHHorQ.pdf" file.
What would be needed to recover the full relaxation matrix - mainly the exact cross-relaxation rates exactly? Would be such an effort useful?
040 - structure of the protein-ligand complex
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 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 (
- cd ~/exercises/040_getCommplexUseProteinLigandDistancesWithoutCorrection/000_cyana
- 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
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- What is the difference WRT 040 ?
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- download
- exercises.tgz, for example, to Documents, and unpack them (in console/terminal, execute: cd Documents; tar xzvf exercises.tgz).that, see first the .upl files, only the distances between protein and ligand:
- cd ~/exercises/045_getProteinLigandDistancesExactAndFixedWeights
- head PxLrewight.upl ../040_getCommplexUseProteinLigandDistancesWithoutCorrection/PxL.upl
- see then the resulting protein-ligand structure
- cd 000_cyana
- remove the Q atoms in demo.pdb as before and see in VMD
- 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
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Prepared by Dr. Jiří Mareš,shaped by discussion with Prof. Julien Orts, Florian Wolf M.Sc. and other members of the research group (https://bionmr.univie.ac.at/people/)
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