C581: Molecular Graphics Workshop Instructions, Fall 1997

Part 1: Getting Started

1. Login to the workstation as "C581" using the password distributed in class (note that UNIX is case-sensitive)
   
   
2. Open a UNIX window (shell) by clicking on Desktop-Unix Shell with left mouse button (LMB). A prompt should appear in the window. You can open as many independent windows as you want this way.
   
   
3. Type "pwd" to see the name of the present working directory and "ls" to list the files and subdirectories in that directory.
   When you first log in the present working directory should be /tmp_mnt/ruser/instruct1/C581.
   
   
4. Type "jot il8.pdb" and spend a couple of minutes browsing throught this pdb file which encodes the average minimized NMR structure of the interleukin-8 dimer. The dimer is two-fold symmetrical and the monomers (each containing 72 residues) are labeled A and B. Exit from the jot editor when you are done.
   
   Make a subdirectory for yourself called lastname_firstname (e.g. my directory would be Stone_Martin) by typing "mkdir lastname_firstname".
   Change to your directory by typing "cd lastname_firstname".
   
   
5. Invoke the modeling program by typing "insightII"
   Wait a minute or so for Insight to start up.
   
   
6. Take a few minutes to familiarize yourself with the layout of the Insight window. In particular, note the following:
   • All selections are performed by SINGLE CLICKING with the mouse. DOUBLE CLICKING can cause problems/confusion - try to avoid it!
   • There is a list of menus along the top
   • Icons on left side of window - these are shortcuts to come of the menu options
   • Command line just below graphics window - this can be used to type in commands instead of using the menus.
   
   
7. Click on the "dot in a square" on the upper right hand edge of the Insight window. This should iconize the window (useful if you want to see your other windows) To get back the full window, click on the iconized window.
   
   
8. Click on the mortarboard icon (pilot tutorials). This gives you a list of available tutorials that may be helpful when doing your Pet Protein Assignment. Do NOT spend much time looking at these now.
   
   

Part 2: Alpha Helix Demo

1. Molecule-get
   Choose pdb, user, click in file name box
   In parameters window, go up and down directory tree to find correct file
   In this case you will need to go up to the parent directory (../)
   Select the file (il8.pdb) - it should appear in file name box
   Execute and wait for molecule to appear.
   Hit cancel to remove the dialog box.
   
   
2. Take a few minutes to explore the ways you can rotate and translate the molecule using the mouse. See also the mouse pad, which lists what happens when you click and hold different mouse buttons.

   The x-axis is horizontal and the y-axis is vertical in the plane of the screen
   The z-axis comes straight out of the screen.

   • Left mouse button - used for selecting molecules/atoms
   • Middle mouse button - translation in xy plane
   • Right mouse button - rotation about x or y axis
   • Left AND Right mouse buttons simultaneously - rotation about z-axis
   • Left AND Middle mouse buttons simultaneously - translation along z-axis
   • Middle AND Right mouse buttons simultaneously - zooms in and out (changes "world scale")
   The part of the molecule that we see is determined by the thickness along the z-axis of a viewing "slab". It is easiest to see this from a side view. To get a side view, click on the icon containg a face profile. Now explore the mouse controlled molecule movements again.

3. At the command line type "colat" to color the atoms according to atom type:
   blue=nitrogen; red=oxygen; green=carbon; white=hydrogen; yellow=sulfur
   
   
4. Molecule-display
   Select "only" and then in the parameter window select subset and click on IL8$HELIX
   The display should switch to only showing the apha helix of one of the monomers
   Take a few minutes to rotate the helix around and study its structure
   Note the relative positions of polar and hydrophobic sidechains. Is this an amphipathic helix? Can you guess which edge of the helix is on the interior of the protein?
   
   
5. Molecule-ribbon, execute. A ribbon should appear that traces the backbone of the helix
   
   
6. Measure-Hbond, execute should display the H-bonds of the helix. Note their orientation relative to the helix axis
   
   
7. At the command line type "delete *" to delete the whole molecule from Insight's memory.
   
   

Part 3: Beta Sheet Demo

1. Recall il8.pdb as above
   
   
2. Molecule-display
   Toggle "only" then in molecule spec box, type "IL8:A24-A30,A36-A43,A46-A49" and hit execute. This should display only the three beta strands of monomer A.
   
   
3. molecule-color
   In molecule spec box, type "IL8:A36-A43", click in the color box and select a color.
   Hit 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
   In molecule spec box, type "IL8:A24-A30,A36-A43,A46-A49" and hit 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
   
   
7. At the command line type "delete *" to delete the whole molecule from Insight's memory.
   
   

Part 4: Tertiary Structure and Disulfide Bonds

1. Recall il8.pdb as above
   
   
2. Molecule-display
   Toggle "only" then in molecule spec box, type "IL8:A*" and hit execute.
   
   
3. Molecule-display
   Toggle "off", then under atom set, select sidechain. Execute.
   This should leave only the backbone.
   
   
4. Molecule-ribbon
   Execute.
   Rotate the molecule around to observe the tertiary fold and the relative positions of the alpha helix and beta sheet.
   
   
5. Molecule-display
   Toggle "only", then in molecule spec box, type "IL8:A7,A9,A34,A50".
   Execute to include in the display the sidechains of the four Cys residues.
   
   
6. At the command line type "colat" to color the Cys residues according to atom type.
   
   
7. Molecule-render. Toggle CPK on. Execute.
   The Cys residues should now appear as CPK space filling representations. Note the sizes of the sulfur atoms.
   
   
8. At the command line type "delete *" to delete the whole molecule from Insight's memory.
   
   

Part 5: Quaternary Structure and Annotations

1. Recall il8.pdb as above
   
   
2. Molecule-color, then type "IL8:A*"in molecule spec box, select a color, and hit execute.
   
   
3. Molecule-ribbon
   Select IL8 (i.e. the whole dimer), and hit execute.
   
   
4. Molecule-display
   Toggle "off", and hit 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 sides of the sheet? Can you identify the two-fold axis of symmetry?
   
   
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.
   
   
6. User-Annotate
   This menu allows you to add several different types of annotations. We will use text annotation 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 don't like the annotation, click on Undo, and try it again.
   
   
8. When you are done, keep this structure up ready to save it in a folder in the next part of the workshop.
   
   

Part 6: Saving and Retrieving Insight Folders

1. File-Save Folder.
   Make sure the Save Object Window contains a "*" indicating that everything 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. e.g if I want to save a folder called "newfolder", the name that will end up in the Folder Name window will be "/tmp_mnt/ruser/instruct1/C581/Stone_Martin/newfolder".
   Hit 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 check that your folder has been saved, you could go to your directory in another UNIX shell and 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 into Insight and at the command line type "delete *" to delete everything from Insight's memory.
   
   
4. File-Restore Folder
   Click on the folder name 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. When you come to do your Pet Protein Assignment you will want to store your pdb file and a few .psv folders in your directory.
   
   

Part 7: Finishing Up

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 necesary otherwise we will quickly run out of disk space!!
   
   
3. Move the cursor into the background (outside of any shells) and drag down the right mouse button to log out. Select yes to confirm the logout.
   

For more tutorials on various aspects of molecular modeling, link to C687 (Molecular Modeling seminar class) tutorials