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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 ~/exercises/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 ~/exercises/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 ~/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.
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