Hands-on exercises on solvent models & electrostatics EMBnet - Molecular Modeling Course 2005

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1 Hands-on exercises on solvent models & electrostatics EMBnet - Molecular Modeling Course 2005 Exercise 1. The purpose of this exercise is to color the solvent accessible surface of a protein according to the electrostatic potential calculated by solving the Poisson Boltzmann equation using the UHBD program. The UHBD program can calculate the electrostatic potential on points of a 3D grid surrounding the protein. This grid can be saved in binary format and read by the chimera program. For license problem, the grid has already been calculated and is provided as model.grd. For information, the UHBD input files are given in the uhbd-input directory. The system under investigation is the ribonuclease T1 complexed with the guanosine 2',3' cyclic phosphothioate (1GSPexercises.sxw in the PDB). The electrostatic potential has been calculated for the protein alone, according to the radius and charges of the CHARMM22 forcefield. The ligand is represented together with the protein in order to see how its polar functions fit with the protein electrostatic potential. Here are the commands: In a terminal. Go into the exercise directory and launch the chimera software:dyna-eps1.inp > cd ESP > chimera model.pdb model.pdb contains the coordinates of ribonuclease T1. Open the command line of chimera: Favorites/Command Line Hide protein bonds Select/Select All Action/Atom-Bonds/hide Load the ligand File/Open Select PDB in "File type" Select ligand.pdb in the dialog box Open Select ligand and show in colored ball and stick Select/Chain/L - 1 -

2 Action/Atom-Bonds/ball&stick Action/Color/By element Show several interesting residues In the command line: select :38.P,40.P,43-46.P,77.P,92.P,98.P Action/Atom-Bonds/show Action/Atom-Bonds/ball&stick Action/Color/By element Select Action/Color/surfaces Action/Color/all colors/white Represent the surface of the protein Select/Chain/P Action/Surface/Show Load UHBD grid File/Open Select UHBD grid in "File type" Select model.grd in the dialog box Open Unshown Color Protein surface according to the ESP Tools/Surface-Binding analysis/electrostatic surface coloring Color surface="msms main surface of model.pdb" Color scheme="3 color symmetric" Color at data value+/- ="10" Color - 2 -

3 Have a look. The binding pocket is mainly defined by residues Tyr38, His40, Asn43, Asn44, Tyr45, Glu46, Arg77, His92 and Asn98. See their influence on the electrostatic potential at the surface of the protein. See how the ligand chemical functions fit to the protein electrostatic potential. The surface of the protein may be hidden or made more transparent to facilitate the comprehension of the interactions. Exercise 2. The purpose of this exercise is to achieve short MD simulations of a small protein, i.e. the heatstable enterotoxin produced by enterotoxigenic Escherichia coli (1ETL in PDB), using different schemes for the electrostatic interactions. Three MD simulations are performed in vacuo using a dielectric constant of 1 and a 12 Å cutoff, with a distance dependent dielectric constant =4r, and finally using the GB-MV2 generalized Born model with no cutoff. Another MD simulation is performed with explicit solvent molecules, in stochastic boundary conditions. The root mean square deviation (RMSD) between the protein conformation along the trajectories and the X-ray structure is calculated for the four simulations. > cd 1etl >./charmm < dyna-eps1.inp> dyna-eps1.out >./charmm < dyna-eps4.inp> dyna-eps4.out >./charmm < dyna-gb.inp> dyna-gb.out >./charmm < dyna-sbc.inp> dyna-sbc.out >./charmm < rmsd-eps1.inp > rmsd-eps1.out >./charmm < rmsd-eps1.inp > rmsd-eps1.out >./charmm < rmsd-gb.inp > rmsd-gb.out >./charmm < rmsd-sbc.inp > rmsd-sbc.out IMPORTANT: There is perhaps not enough time to perform the simulation with explicit solvent. The trajectory and the corresponding files will thus be provided. Have a look at the dyna-sbc.inp and dyna-sbc.out files to see what is done. Several files are generated: traj/eps1.dcd traj/eps4.dcd traj/gb.dcd traj/sbc.dcd coor/eps1.crd and coor/eps1.pdb - 3 -

4 coor/eps4.crd and coor/eps4.pdb coor/gb.crd and coor/gb.pdb coor/sbc.crd and coor/sbc.pdb rmsd-eps1.dat rmsd-eps4.dat rmsd-gb.dat rmsd-sbc.dat Have a look at the input files to guess what these files correspond to. NOTE: It is possible to have a look at some results, like an MD simulation, while the rest is still being calculated. For example, to look at the MD simulation done with a a dielectric constant of 1: > vmd data/1etl.pdb In VMD: Display/orthographic Graphics/Representation Create Rep Select the second representation Drawing Method: Hbonds Line Thickness: 4 File/New Molecule Load files for: 0: 1etl.pdb Filename: traj/eps1.dcd Load Close the New Molecule dialog box - 4 -

5 How many Hbonds are present in the X-ray structure? How many exist at the end of the MD simulation? Follow their appearance during the MD simulation. Do the same for the other simulations. For clarity, in the case of the MD simulation with explicit solvent, it is preferable not to show the water molecules when looking at the hydrogen bonds within the protein (Please ask if you don't know how to do it). Do you see a difference? When all the calculations are finished, have a look at the RMSD along the trajectory. > xmgrace -p rmsd.param rmsd-eps1.dat rmsd-eps4.dat rmsd-gb.dat rmsd-sbc.dat Compare. Exercise 3. The purpose of this exercise is to study rapidly the separation of two insulin monomers starting from the insulin dimer, including or not the solvent effect. Have a look at the insulin structure using molmol: > cd insulin-dimer > molmol dimer.mml Starting from the X-ray structure of an insulin dimer (4INS in the PDB), the two monomers are separated by successive small translations of the second one. After each small translation, the energy of the system is calculated. The calculation is done twice: in vacuo with a dielectric constant of 1 and no cutoff, and using a GBSA solvent model. >./charmm < no-solv.inp> no-solv.out >./charmm < gbsa.inp> gbsa.out - 5 -

6 Two files are generated that contains the energy of the system as a function of the total translation: trans-ene-nosolv.dat and trans-ene-gbsa.dat. For trans-ene-nosolv.dat : column 1: translation column 2: total energy (including bonded terms) column 3: vdw energy column 4: electrostatic energy For trans-ene-gbsa.dat : column 1: translation column 2: total energy (including bonded terms) column 3: vdw energy column 4: electrostatic energy column 5: electrostatic free energy of solvation column 6: nonpolar solvation energy Look at the energies as a function of the total translation and compare the two experiences. > xmgrace -p nosolv.param -nxy trans-ene-nosolv.dat > xmgrace -p gbsa.param -nxy trans-ene-gbsa.dat What is the main difference between the two total energy curves (for the GBSA curves, you will need to zoom in the -1200/-1500 kcal/mol range)? What effects are responsible for this difference? What is the more realistic model? Exercise 4. The purpose of this exercise is to compare solvation energies calculated using different implicit solvent models: the Poisson Boltzmann equation and the GB-MV2 Generalized Born model. The solvation energies were calculated for several conformations of the insulin dimer, extracted from an MD simulation with explicit solvent. This MD simulation will not be described here. Two programs are used to solve the Poisson Boltzmann equation: UHBD and the PBEQ module of CHARMM. The solvation energies were already calculated with the UHBD program and are provided in the uhbd.dat file. In fact, since we use a null salt concentration, we actually solve the Poisson equation. > cd PB-GB >./charmm < GBMV2.inp > dyna-eps1.out >./charmm < PBEQ-VDW.inp > PBEQ-VDW.out >./charmm < PBEQ-MS.inp > PBEQ-MS.out The PBEQ-VDW.inp and PBEQ-MS.inp input files solve the Poisson equation using two different definitions of the molecular volume: the volume defined by the van der Waals or the molecular surfaces, respectively. UHBD uses the molecular surface to define the molecular volume

7 Compare the CPU time required to do these calculations (last lines of the GBMV2.out, PBEQ- VDW.out and PBEQ-MS.out files). The UHBD calculations takes about 12 minutes on a Pentium IV 3.2 Ghz. The electrostatic solvation free energies calculated by the different methods are now stored in the gbmv2.dat, pbeq-vdw.dat, pbeq-ms.dat and uhbd.dat. To compare the results, we can put all these data in a single file and open it with xmgrace: > paste uhbd.dat pbeq-vdw.dat pbeq-ms.dat gbmv2.dat > all.dat Load the file in xmgrace: > xmgrace -p compare.param -nxy all.dat & Then calculate the regression between the UHBD results on one hand and the three other methods on the other hand. In xmgrace: Data/transformations/Regression... Select set S0 (PBEQ-VDW) Accept Note the equation of the regression (slope and intercept) and the correlation coefficient. Do the same for the PBEQ-MS (set S1) and GB-MV2 results (S2) Compare