Text-based measurement of atomic and molecular properties.

1. Start InisghtII and get the PDB file /ruser/instruct1/stone/C687/homology/3znf.pdb. Edit this structure so that the two cysteines form a disulfide bridge. Fix, Fix, Fix your potentials, partial charges, and formal charges.
2. List properties of the molecule
   • Click on Molecule/List, and list the extents of the molecule The extents are the X, Y, and Z dimensions of the molecule (the orientation of the X, Y, and Z axes are defined in the WORLD coordinate frame) When is this information important???
   • List the chralities of the molecule. Some chiralities of the CA (alpha carbon) atoms are "R", while most are "S". Investigate why this is so.
3. Using the Biopolymer module, Click on residue/list. First try this with ONLY the name of the molecule (with no ":" characters).
4. click on protein/list. List various properties of the protein. Which propertie(s) are listed "incorrectly"? Why is the program "incorrect"?
5. click on atom/list. Click on ExDetails and click execute. Two lines for each atom will be printed in the textport. Basic information, such as the name of the group to which the atom belongs, atom type, X,Y,Z coordinates, and which atoms are connected to the atom are listed for each atom (on the first line). Extra details, including the potential atom types, partial charges, and formal charges are listed on the second line for each atom. Notice the general range of partial charges.

   The N-terminal N itrogen has a formal charge of +1, but the partial charge of this atom is -0.500! Why??? The anser is because this this atom is designated as the SWITCH atom for that group. The sum of the potential charges of the entire group should be the same as the formal charge. Therefore, if you want to change the formal charge on an atom, you must also be sure that the sum of the partial charges of the group also equal the formal charge.

6. Blank on the protein (use object/blank)
7. Restore the folder /ruser/nmrsg1/mpagel/C687/dna.psv This is a duplex of DNA (with only G_C bases, in that order) with 8 A_DNA_duplex base pairs, 8 B_DNA_duplex base pairs, and 8 A_RNA_DNA_duplex base pairs.
8. OPTIONAL STEP (DON'T BOTHER DOING THIS TODAY. INSTEAD, JUST READ THROUGH THIS STEP AND GO ONTO THE NEXT STEP): The DNA duplex that you just restored consists of TWO molecules. Click on Object/list to list the names of these molecules, which will be "your~molecule~name_1" and "your~molecule~name_2". If you "connect object" and connect to one of the two strands, and you move the object, you will only move one of the two strands---you will lose the double helix conformation!. There are two methods that you can use to avoid this: A. While the double helix consists of two non-covalently-bound molecules, the two molecules are also defined as an assembly. Click on Assembly/list to list this DNA assembly. To move BOTH DNA strands together, Click on Transform/connect and connect to the DNA assembly (NOT one of the two molecules). B. To merge the two molecules together WITHOUT making a covalent bond between the molecules, click on modify/merge, and merge one molecule into the other. If you desire, you can rename the molecule using Object/rename. Then you can move the entire molecule with no problems.
9. Change the color of one strand to crimson (red) and the other strand to cream (white). Change the 5' end of the red strand to green and the 3' end to yellow.
10. Click on Nucleotide/measure. Define the 5' end (green residue) and the 3' end (yellow residue). Measure from the green residue to the yellow residue (what happens if you select a different region to measure?). Click Execute. Click on Texport On.
11. What is all of this junk in the window??? Scroll around the window and compare these vaules with the structure on the screen. What geometrical differences exist for A-DNA, B-DNA, Z-DNA, and a DNA/RNA hybrid? How do the parameters compare for nucleotides at the interfaces/ends of the A_DNA, B_DNA, Z_DNA, and DNA/RNA regions vs the nucleotides in the middle of these regions?
12. Delete the DNA, Blank "off" the zinc finger structure.

Graphics-based measurement of atomic properties

1. Color the atoms of the protein by their partial chargei:
   1. Click on molecule/color, set up the menu to color all atoms of the protein.
   2. Set Color Method to property. property_level should be "atom".
   3. Set Property to Partial_charge and Spectrum_Name to CHARGE_SPECTRUM. The CHARGE_SPECTRUM is a predefined scale that will color your atoms from red (-0.5) to blue (+0.5) depending upon their partial charge value. Note that there are other pre-defined spectra.
   4. Click on Execute, and the atoms will change from red to blue. The spectrum scale will also appear. This spectrum scale is automatically named MOL_SPECTRUM.
2. Editing Sepctra
   Click on Spectrum/edit. Select MOL_SPECTRUM and click OK. Edit the properties of this spectrum:
   1. Change the Over Range Color to green and Under Range Color to yellow. Recolor your molecule (Spectrum_name is MOL_SPECTRUM). Idtentify if any atoms have partial charges that are greater than the maximum (+0.5) or less than the minimum (-0.5) values of the spectrum.
   2. Edit MOL_SPECTRUM and change label options, scale options, the minimum and maximum colors. Recolor your molecule.
   3. Delete all spectra using object/delete.
3. Create your own spectrum
   Let's make a spectrum based upon sequence number, and then make a colorful ribbon.
   1. Click on Spectrum/edit, and click on "New Spectrum". Enter a spectrum name. Set minimum value to 1 and maximum value to 30 (the zinc finger has 30 residues). You MUST first type in the maximum value, then type in the minimum value. If you switch this order, the program will NOT set the minimum and maximum to the new values, since the minimum value must NEVER be equal to or greater than the maximum value. Click OK.
   2. Verify that your minimum and maximum values are OK.
   3. Split your spectrum (click on split). Define the maximum value of subrange #1 to be white, and the maximum value of subrange 1 to be 15. Define the minimum value of subrange #2 to be 15 and the minimum color to be white.
   4. Color your molecule:
      color_method = property
      property_level = monomer
      property = sequence_number
      spectrum_name = your new spectrum
   5. Make a solid rectangular ribbon
4. Delete everything EXCEPT the zinc finger structure. Save your zinc finger structure as a folder. Quit InsightII.

1. The Solvation module is currently licensed only on chemvgx and splatter. Therefore, when working on other machines it is necessary to run the program remotely on chemvgx or splatter, then display on your local monitor. Half of the class should choose each machine. e.g. To run remotely on chemvgx,
   xhost chemvgx
   telnet chemvgx (then login)
   cd to appropriate directory
   insightII
   You can also log onto splatter using your OWN account, with your OWN home directory.
   However, your accounts on splatter will be removed after the tutorial; please use chemvgx in the future.
2. Restore your zinc-finger folder.
3. Choose the Solvation module and take a few minutes to browse through the menu options.
4. Set up your system (click on setup/system, and select your protein).
5. Set up your parameters:
   Surface area only
6. low resolution (just to limit the timre required for the calculation for this tutorial) If you want to calculate surface areas for your research, choose a higher level of resolution. (Regular or High resolution? Rule #1 of molecular modeling: Test both and see what happens!)
   Output level = atom
7. To run, click on solvation_run/run.
8. wait about 1-3 minutes for the calculation to proceed. Meanwhile, think of a question that will stump Martin, Marty, or Brandt.
9. When the calculation is finished, wait until the table is finished being created and displayed. Then delete this useless box: click on the square in the upper left corner of this window, then select close.
10. In a UNIX window, list the files that were created. type more your_molecule#.hydro_log". For example, my file is named solv_znf1.hydro_log, so I typed more solv_znf0.hydro_log.
11. Read through this file and identify which residues are solvent exposed and which are buried in the protein structure. Which atoms of solvent-exposed residues are buried?

Free energy of SOLVATION

Drug designers often like to calculate the free energy of solvation from octanol to water. Octanol is assumed to approximate the environment inside the protein. Therefore, the free energy of solvation measures the non-specific "affinity" of the drug for the protein binding site vs the aqueous solution around the protein. How can you use this really cool calculation in your individual project? Discuss this with Martin or Marty.

Some chemists consider the hydrophobicity to be the free energy of solvation from a vacuum to water, while others consider the free energy of solvation from octanol to water to be a more accurate measurement. What do you think?

1. If you are not running the solvation module from chemvgx or splatter, see the instructions in the Solvent Accessible Surface Areas section of this tutorial.
2. Using Builder/fragment/get, get a leucine amino acid and a lysine amino acid as 2 molecules (do NOT connect them with a bond).
3. Click on Forcefield/select and select the CFF91 forcefield. The Eisenberg_McLachlan Octanol_Water_Model is based upon the (electrostatic and van der Waals) CFF91 parameters---other forcefields will NOT work!
4. Click on Forcefield/select and fix the atom potentials, fix the partial charges, and fix the formal charges of the two amino acids. While you aren't using the zinc finger for this part of the tutorial, you should also fix/fix/fix the zinc finger (or you can change the forcefield back to CVFF for the electrostatics calculation).
5. Perform the following measurement two times, once for each amino acid:
   1. In the solvation module, Set up your system (click on setup/system, and select the amino acid).
   2. Set up parameters:
      ONLY select the Octanol_Water_Model: Eisenberg_McLachlan protcol
      low resolution (just to limit the timre required for the calculation for this tutorial) If you want to calculate surface areas for your research, choose a higher level of resolution. (Regular or High resolution? Rule #1 of molecular modeling: Test both and see what happens!)
      Output level = atom
   3. To run, click on solvation_run/run.
   4. wait about 10-30 seconds for the calculation to proceed. Meanwhile, answer a question that Martin, Marty, or Brandt will ask you to try to stump you.
   5. When the calculation is finished, wait until the table is finished being created and displayed. Then delete this useless box: click on the square in the upper left corner of this window, then select close.
   6. In a UNIX window, list the files that were created. type more your_molecule#.hydro_log". For example, my file is named solv_leu2.hydro_log, so I typed more solv_leu2.hydro_log.
   7. Read through this file and identify which residues are solvent exposed and which are buried in the protein structure. Which atoms of solvent-exposed residues are buried?

Electrostatics

This tutorial will generate a 3D grid of points centered on your molecule. The electostatic potential felt by each grid point from the partial charges of each atom is then calculated. Finally, the surface of the protein is created and colored based upon the electrostatic values.

How the electrostatic potential calculation is accomplished:
Consider two points in space that are fairly close together, both of which have a partial charge:

1. One point "radiates" it's electrostatic potential, and the other point "feels" this potential. The second point "reacts" (via dipolar reorientation and electronic polarization) to this potential and the electrostatic potential of the second point changes in value.
2. The second point, with it's new value, "radiates" it's potential, and the first point "feels" this potential. The first point "reacts" to this potential, and the electrostatic potential of the first point changes in value.
3. Since the first point has changed it's electrostatic potential value, the calculation returns to step 1 with this new value of the first point. This calculation loop is repeated until the changes in the electrostatic potential values are very small.

Each grid point is calculated based upon it's "reaction" to the values of the 6 neighboring grid points. This causes two problems:

• Since we can't calculate an infinite grid, we must have a boundary to our grid. Points at the boundary of the grid only have 3, 4, or 5 grid points, causing innacuracies. (Which grid points have 3 neighbors? Which have 4? Which have 5?). To reduce this error, we can make a grid with boundaries that are far from the molecule, which resides at the center fo the grid.
• Since we are essentially extrapolating the electrostatic potential values from one grid point to another, the grid points should be very close together (i.e., the grid resolution must be very small) to avoid any "extrapolation" or "round-off" error.

1. Calculate a very large grid with only average resolution, as described below.
2. Then repeat the calculation, select a smaller grid (e.g., a grid that is not much larger than the size of the protein), set the Boundary to "focussing", and set the focussing grid to the grid that you calculated in step 1.

The tutorial performs these steps:

• set up the system
• create a grid
• calculate the electrostatic potential at each grid point
• create a surface of the zinc finger
• color the surface based upon the grid points nearest to the surface.

1. If you are not running InsightII from chemvgx or splatter, see the instructions in the Solvent Accessible Surface Areas section of this tutorial.
2. If you don't have the zinc finger structure, get the PDB file /ruser/instruct1/stone/C687/homology/3znf.pdb. If you are not sure if the zinc finger structure has the correct atom potential types, partial charges, or formal charges, then select a forcefield, and fix/fix/fix your potentials.
3. Select the DelPhi tutorial.
4. Under Setup/Boundary, select Full coulombic
5. Under Setup/Grid:
   select display_grid
   Grid Center = Molecule_Region
   Molecule Region = your molecule
   Grid Size = Border_Space
   Border Space = 10
   Grid Resolution = Point_Spacing
   Angstroms/Grid Pt = 1
   
6. View the parameters under Setup/Solute, Setup/Solvent. These default parameters are set up for a typical protein solute in a physiological solvent.
7. Click on Setup/Initialize, and initialize the calculation with default parameters.
8. Click on Run_DelPhi/Run, and run your calculation in the background.
9. The calculation can take 3-10 minutes. Once the calculation is finished, a box will appear to notify you.
10. To generate a surface of your protein, click on Molecule/Surface. Create a Solid Connolly surface with an atom radius of 1.4 and an atom radus incr of 0.00. For this tutorial, select a low-resolution surface quality.
11. Wait a few minutes for this surface to be calculated.
12. Color the surface of the molecule, with Color Method = Grid, spectrum Name = CHARGE_SPECTRUM, and Scalar Grid Name = the name of the grid you calculated.