Names: ______________________

______________________

1. Login to the workstation as "C581" using the password discussed in class (note that UNIX is case-sensitive).
2. If there is not a window already open, go to "Desktop" and select "Open UNIX Shell." A prompt should appear in the window. Any number of independent windows can be opened this way.
3. Type "pwd" to see the present working directory. This directory should be

/tmp_mnt/ruser/instruct1/C581.

Type "ls" to list the files and subdirectories in this directory.

4. Make a subdirectory for yourself called lastname_firstname (e.g. Gupta_Sonia) by typing "mkdir lastname_firstname". Note: Any work you do on these computers must be done under either your own or your partner’s directory.

3. Once Netscape is open, go to "Bookmarks" and select "PDB WWW Home Page."
4. Under "Search", select Search Lite. A new page will appear.
5. Next, browse for a protein. To do this, type the PDB code of the protein you want to find in the "Keyword" box (e.g. 1dgc). Click on "Search". Another page will appear that will list some files containing your protein. Select one of these and then click on "{EXPLORE}". A screen will appear that will give you more information about your protein. To save this file, so that it can be opened in the Insight2 program, choose the "Download/Display File" option from the panel on the left. Scroll down to "Download the Structure File". Click on the "X" marked for the "PDB file format" in the "none" compression mode. Before you click on OK, make sure that the file is being saved into your directory. Note the pdb code (e.g. 1DGC.pdb).
6. Exit Netscape. Now, you can jot the file, like before, to browse the file and look for interesting features. Type "jot" and then the name of the pdb file (e.g. jot 1DGC.pdb) in the UNIX shell. Take a few minutes to browse through the file and then exit the jot window. Look for the title of the article, the method used to solve the structure, the journal and the authors (under JRNL TITL). Also, look for additional references (in the REMARK section). Browse through the experimental details. Finally, browse through the SEQRES section to look at the residues that comprise your protein, their numbering scheme, and the letter of the strand they are located in.

1. Start the modeling program by typing "Insight2" in the UNIX shell. It will take a couple of minutes for the program to load.
2. Take a few minutes to become familiar with the Insight window. Be sure to note the following:

• Avoid DOUBLE CLICKING because this can cause problems/confusion. Instead, use SINGLE CLICKING to make all selections.

• Across the top of the window is a list of menus.
• Along the left side of the window, are icons. These are shortcuts to some of the menu options.
• The command line is located directly below the graphics window. This can be used to type commands instead of using the menus.

1. Go to "Molecule" and choose "Get." Make sure "PDB" is selected in the "Get File Type" box. Because you are still in your directory, you will need to single click on the parent directory (../) in the "Files" box. Select the file 1il8.pdb. Click on Execute. Click on Cancel to remove the dialog box once the molecule has appeared.
2. At first sight, this molecule appears to be a big mess. However, it is really two asymmetric molecules. One is Strand A and the other is Strand B. Next, we will look at some different ways that you can use to specify which atoms or groups of atoms should be displayed. These techniques can be applied to control other properties also, such as color etc.

 

Following are some examples of how to specify atoms:

IL8 specifies all atoms in the molecule labeled IL8

IL8:A* specifies all atoms in molecule A in the pdb file containing the IL8

structure

IL8:B* specifies all atoms in molecule B in the pdb file containing the IL8

structure

IL8:A25 specifies all atoms in residue number 25 in molecule A

IL8:A25-A32 specifies all atoms in residues 25 to 32 in molecule A

IL8:A25,A30,A35 specifies all atoms in residues 25, 30, and 35 of molecule A

IL8:Cys specifies all atoms in all Cys residues in either molecule A or B

IL8:B50:N specifies the amide nitrogen in residue 50 of molecule B

IL8:B50:CA specifies the alpha carbon in residue 50 of molecule B

IL8:B50:C,N,HN,CA,HA,CB,HB1,HB2

specifies all of the listed atoms in residue B50

 

If you are unsure about how to specify an atom, cancel all menus, and then click on the atom of interest. The atom’s specification will appear in the panel below the command line. Remember that doing this selects the atom. Click in a clear space to deselect the atom.

 

3. By clicking on "Molecule" and selecting "Display," you can choose to display certain subsets of atoms. (You can also use the ON/OFF icon to do the same thing). Under this menu you can choose an atom set and indicate whether you want it turned ON, OFF, or to be the only one set ON. The atom set can be picked from the "Atom Set" menu or typed directly into the "Molecule Spec" box using the above formats. Spend a few minutes trying the various options to display various sets of atoms.

 

4. "Delete" this molecule and "Get" it again. Type "delete *" at the command line to delete the entire molecule from Insight’s memory.

 

5. Next, we will look at only the alpha helix portion of this molecule. Go to "Molecule" and select "Display." Click on "Only" in the "Display Operation" box. Type "IL8:A56-A71" in the "Molecule Spec" box. Click on Execute. This will display only the alpha helix portion of the molecule. Click on Cancel to remove the dialog box.

 

6. Draw a ribbon that traces the backbone of the helix. To do this, go to "Molecule"-"Ribbon" and then click on Execute. Make sure "Create" is selected under "Ribbon Operation".

 

 

 

 

 

 

 

 

 

 

7. Take a few minutes to learn the various ways to rotate and translate the molecule using the mouse.

• Left mouse button – used for selecting molecules/atoms

• Middle mouse button – translation in the xy plane
• Right mouse button – rotation about the x or y axis
• Left AND Right mouse buttons simultaneously – rotation about the z-axis
• Left AND Middle mouse buttons simultaneously – translation along z-axis
• Middle AND Right mouse buttons simultaneously – zooms in and out

1. Rotate the helix around. View the helix end-on and side-on.
2. Color the hydrophobic sidechains yellow and the hydrophilic sidechains dark blue. For this, select "Molecule"-"Color". Use the residue specification format you saw in step 4. What do you notice about the distribution of the hydrophobic and hydrophilic groups? Is this helix amphipathic?
3. Remove the ribbon and turn off the sidechain display. To remove the ribbon, go to "Molecule" and select "Ribbon". Click on "Delete" in the "Ribbon Operation" box. Click on Execute. To remove the sidechains, go to "Molecule" and select "Display". Choose "Off" and then select "Sidechain" under "Atom Set". Execute. This will leave only the backbone.
4. Measure the H-bond distances. "Measure"-"HBond" and Execute should display the H-bonds of the helix.

To measure the length of a H-bond, go to "Molecule"-"Distance" and then click on the atoms of interest. What is the range of H-bond distances in this helix?

Which groups are H-bonded in an alpha helix?

How many residues are there per turn of an alpha helix?

5. Measure the dihedral angles (f and y ) in the helix. To do this, select "Measure" and then choose "Dihedral". Select the 4 atoms in the angle by clicking on them. What are these angles?
6. Delete this molecule.

1. "Get" the 1il8.pdb molecule again. You do not have to click on the parent directory (../) this time because you are already in this directory.
2. "Molecule"-"Display".

Click on "Only." In the "Molecule Spec" box, type "IL8:A24-A30,A36-A43,A46-A49" and click on Execute. This should display only the three beta strands of monomer A.

3. "Molecule"-"Color"

In the molecule spec box, type "IL8:A36-A43", click in the color box and select a color. Click on Execute. This should display the middle strand in a different color. Color one of the other strands as well, if you want.

4. "Molecule"-"Ribbon"

Make sure "Create" is selected in the "Ribbon Operation" box. In the "Molecule Spec" box, type "IL8:A24-A30,A36-A43,A46-A49" and click on Execute.

5. Take a few minutes to rotate the sheet around and study its structure. Note that it is twisted rather than flat.
6. "Measure"-"HBond", Execute should display the H-bonds of the sheet. Note their orientation relative to the "plane" of the sheet.

Remove the ribbon and turn off the sidechain display.

What is the range of H-bond distances in this sheet?

What is the translation per residue in the beta strands?

 

7. Measure the dihedral angles (f and y ) in one of the strands. To do this, click on "Measure" and choose "Dihedral". Next, select the 4 atoms in the angle. What are these angles and how do they compare to those for an alpha helix?

 

 

 

 

8. Delete this molecule.

1. "Get" the 1il8.pdb molecule again.
2. "Molecule"-"Display"

Click on "Only." In the "Molecule Spec" box, type "IL8:A*" and hit Execute. This will display only the A monomer.

3. "Molecule"-"Display"

Click on "Off", select "Side Chain" under "Atom Set" and Execute. This should leave only the backbone.

4. "Molecule"-"Ribbon". Make sure "Create" is selected. Execute.

Rotate the molecule around to observe the tertiary fold and the relative positions of the alpha helix and beta sheet.

5. "Molecule"-"Display"

Click on "Only." In the "Molecule Spec" box, type "IL8:A7,A9,A34,A50".

Click on Execute to include the sidechains of the four Cys residues in the display.

6. Type "colat" at the command line to color the Cys residues according to the atom type. "Colat" colors the atoms according to the atom type: blue = nitrogen; red = oxygen; green = carbon; white = hydrogen; yellow = sulfur; pink = phosphorus.
7. "Molecule"-"Render". Click on "CPK" and then Execute.

The Cys residues should now appear as CPK space filling representations. Note the sizes of the sulfur atoms. Try other "Render Styles" also.

Choose "Lines" as the "Render Style" to do the next step.

8. Measure the dihedral angle around the disulfide bond ("Measure"-"Dihedral"). What is this angle?

 

 

9. Delete this molecule.

1. "Get" the 1il8.pdb molecule again.
2. "Molecule"-"Color". In the "Molecule Spec" box, type "IL8:A*", select a color, and click on Execute.

3. "Molecule"-"Ribbon". Select IL8 (i.e., the whole dimer), and click on Execute.

4. "Molecule"-"Display"

Select "Off" and click on Execute.

This should leave a ribbon representation of the dimer with each monomer colored differently. Spend some time observing the dimer structure and the way that the two monomers pack together. Note the contiguous 6-stranded beta sheet.

Are the two helices on the same or opposite side of the sheet?

What interactions appear to hold the dimer together?

5. Can you identify the two-fold axis of symmetry? Sketch the appearance of the structure and indicate the position of the two-fold axis of symmetry.

1. "Session"-"Quit", then Execute will allow you to exit from Insight.
2. In a UNIX shell type ‘pwd’ to make sure you are in your own directory. Then list the files (ls). Remove any file you no longer need (which should be all files) by typing "rm filename". Remove all the unnecessary files so that we don’t run out of disk space!
3. "Exit" all windows and "Log out".