Names _________________

1. Login using "C581" as the user and use the password supplied in class.
   
2. Open a UNIX shell and change to your directory (cd lastname_firstname).
   
3. Start netscape by typing "netscape" in the UNIX shell.
   
4. Once netscape is open, go to "Bookmarks" and select "PDB WWW Home Page.
   
5. Under Search the PDB:, select 3DB Browser.
   
6. Next, browse for a protein of your choice. To do this, type the name of the protein you want to find in the Keyword box. Scroll down and click on the " Search" box located below the Data Source box. A box will appear that will list some files containing your protein. Select one of these and then click on the "Retrieve Data" box. A screen will appear that will give you more information on your protein. To save this file so that it can be opened in the Insight2 program, choose the [Save to Disk] option under Data Retrieval. Before you click on OK, make sure that the file is being saved into your directory. Note the pdb code (e.g. 1DGC.pdb).
   
7. 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 method that was used to solve the strucutre (under EXPDTA), the title of the article where the structure was solved, the authors, and the journal. 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, the letter of the strand where they are located.
   

1. Start Insight2.
   
2. 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.
   
3. 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, such as color.

   The 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 how to specify an atom, cancel all menus, and then click on the atom of interest. The atoms 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.

    
4. 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 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 approximately 15 minutes trying the various options to display various sets of atoms. You will probably have to delete and get the molecule again in order to go through the various specifications.

5. Also, use the Molecule-color option to color different parts of the molecule. Delete this molecule and "get" it again.
   
6. 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.
   
7. Draw a ribbon that traces the backbone of the helix (molecule-ribbon, execute).
   
8. Rotate the helix around. View the helix end-on and side-on.
   
9. Color the hydrophobic sidechains yellow and the hydrophilic sidechains dark blue. What do you notice about the distribution of the hydrophobic and hydrophilic groups? Is this helix amphipathic?
   
10. 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.

11. Measure the H-bond distances. Measure-Hbond, execute should display the H-bonds of the helix.

    What is the typical H-bond distance in this helix?

    Which groups (and which residues) are H-bonded?

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

    What is the rise per turn of the helix?

    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 as 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. Click on Textport On and then Textport Off to place the UNIX shell behind the Insight window.

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. Measure the H-bond distances.

   What is the typical H-bond distance in this sheet?

   What is the translation per residue in the beta strands?

7. 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" and select side chain under atom set. Click on Execute. This should leave only the backbone.

4. Molecule-ribbon

   Click on 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 in the display the sidechains of the four Cys residues.

6. Type "colat" at the command line to color the Cys residues according to atom type.
   
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.

8. Try some different molecule renderings. Choose "Line," "Ball_and_Stick," and "Stick." Also, take some time to try and color different parts of the molecule.
   
9. Measure the disulfide bond distances. What is this distance?
   
10. Measure the dihedral angle around the disulfide bond (Measure-dihedral). What is this angle?
    
11. 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?

   Can you identify the two-fold axis of symmetry?

   What interactions appear to hold the dimer together?

5. Orient the dimer in a direction that looks aesthetically pleasing and informative. You should be happy that you have the best orientation before you add any annotations. Sketch the appearance of the structure and indicate the position of the two-fold axis of symmetry.
   
6. User-Annotate

   This menu allows you to add several different types of annotations. The text annotation will be used as an example. Choose "text." Under "attach to" it should say "annotation." In the "text" box, type the text you want to add. Click in the X-Coord1 box, then move the cursor off the window and over the protein.

7. Either (a) click the left mouse button when the cursor is positioned where you want the annotation to appear, or (b) click and hold the left mouse button; the annotation will appear; drag the annotation to the desired spot; release the left mouse button. If you do not like the annotation, click on Undo, and try it again.
   
8. When you are done, keep this structure up and ready to be saved into a folder in the next part of the workshop.

1. File-Save Folder.

   Make sure the Save Object Window contains a "*" indicating that everyting should be saved. Click in the Folder Name window, then click on the name of your directory. Click the cursor at the end of the long (full pathname) directory name that appears in the Folder Name window and append to that name the name under which you want to save the folder (i.e. to save a folder called "protein", the name that will end up in the Folder Name window will be "/tmp_mnt/ruser/instruct1/C581/McClain_Diana/protein". Click on Execute to save everything you have in the Insight memory into your folder. Note that a folder is different from a directory. The word "folder" means something different from the way it is used on a Mac or PC.

2. To make sure that your folder has been saved, open another UNIX shell and change to your directory. Type ls to list the files. The folder name has a .psv suffix.
   
3. When you are confident you have saved your folder correctly, go back to Insight and at the command line type "delete *" to delete everything from Insight’s memory.
   
4. File-Restore Folder

   Click on the folder name that you saved and then Execute. The representation that you saved along with all the relevant coordinates, annotations, etc. should be restored to Insight’s memory so that you can continue exactly as you could have before saving the folder.

5. For future assignments, you will want to store your pdb file and a few .psv folders in your directory.
   

1. Session-quit, then Execute will allow you to exit from Insight.
   
2. In a UNIX shell 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". When you are working on your assignment you should always clean up any files that are not absolutely necessary, otherwise we will quickly run out of disk space!
   
3. Exit all windows and log out.