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Remark: CYANA is a proprietary software. For any installation problem, contact Peter Güntert, the author of CYANA.
Task 1 (030) - structure of protein
- 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)
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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.
031 - structure of protein from approximate distances
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.
- 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 -l PxPapp.upl
- Start the structure calculation, or see the ready demo.pdb and demo.ovw file.
032 - ... continuation
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, 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?
034 - ... continuation
In the next simple attempt, we multiply all the distances by a constant factor (1.75) and use again those within 5.5A. 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.
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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 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.
- How would you determine structure of a ligand in the bound state?
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038 - effect of exchange on NOESY spectra (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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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) - 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.
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You saw that the NOESY crosspeak can be in principle recovered exactly even if the atoms, mediating the relayed magnetization transfer, are merged into a single pseudoatom.
- See the time-evolution of the cross-peak (first and last atom) as plotted in "relayedThroughHHorQ.pdf". What does it mean?
- 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.
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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 protein-ligand complex
We have exact distances (assume we are able to obtain them), but we ignore the populations, so the intermolecular calibration is wrong.
- Check the PxL.upl
- What are the effects when calculating the protein-ligand complex?
- When viewing the complex using VMD, in graphical Graphical representation, you can select the atoms of proteins by typing "proteinsprotein" and the ligand as "not protein"
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