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.

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

...

• 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 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.

They are written Upper distance Limit files (here PxP.upl) file, which is used by CYANA.

Intermezzo: CYANA

There is no closed-form formula to calculate the conformation (structure) from a set of distances. The setup starts with defining an energy penalty for every experimental distance not fulfilled by the molecular conformation. These are also called distance restraints. Starting from one chosen conformation, and trying to minimize the structure (using steepest descent or other local method) to fulfill the distances measured by NOE (or any other means) would fail: the structure would end-up in a local minimum. Instead we have to search for a global minimum. A commonly used algorithm for a global minimum is called simulated annealing, where the molecule is heated up such that high energy barriers (due to van der Waals clashes) can be surpassed. By a subsequent cooling, the imposed distance restraints will drive the molecule towards the conformation with minimal violation of the distance restraints. Many attempts will nevertheless end up in different local minima, and hence, only a subset of resulting conformers, the lowest-energy conformers will be likely to represent the global minimum.

...

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, 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.

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.

• Check how many are left, what is the statistics of the structure calculation. And
• visualize the structure.
• Switch again to NewCartoon drawing method. What happens?
• What do you think, were these attempts good enough?

Task 3: Adding the exchange

.
We will investigate the effect of exchange of the ligand between the free state in the solution and bound to the protein.

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?

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. 

...

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.

...

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 

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 population 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 (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 are take into account correctly, so also the intermolecular distances are calibrated correctly.

...