C687: Introduction to InsightII

Part 1: Getting Started

1. In a UNIX shell, cd to appropriate directory.
2. Type insight2 or insightII
3. Wait a minute or so for InsightII to start up.
4. 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!
   • The menus along the top - go through these checking out the various options
   • The Biosym icon in the upper left corner - click to see options for other modules
   • 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.
   • Panel in lower left corner to facilitate molecule rotations/translations etc.
   • Option keys along bottom to perform various useful commands.
5. 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.
6. Click on the mortarboard icon (pilot tutorials). Check out what tutorials are available - these will be very helpful.
7. Go to session-quit to exit from Insight. Confirm exit. It takes a minute for the program to shut down.

Part 2: Getting and Moving Molecules

1. Start up InsightII again.
2. Molecule-get
   Choose pdb, user, click in file name box
   In parameters window, go up and down directory tree to find correct file
   Select file (should appear in file name box)
   If you desire, change the molecule name under Get Molecule to a name that you prefer.
   Execute and wait for molecule to appear.
   
3. Molecule-put Choose pdb
   In file name box, type name of file.
   Execute
   Now do a directory listing in a UNIX shell to see the new file.
   
4. Back in Insight, spend some time exploring 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 (see later)
   • Middle mouse button - trnslation 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.

   Finally, explore using the panels in the lower left corner to effect molecular rotation, translation, world scale, and slab thickness.

5. Type on the command line delete *. This will delete all objects you currently have in InsightII (very useful).

Part 3: Molecular Specifications and Diplaying/Coloring Atoms

Examples of the general format for specifying 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 don't know how to specify an atom, cancel all menus then click (left) on the atom of interest. The atom specification will appear in the panel below the command line. This action selects the atom. Click in clear space to deselect. The atom and molecule labels in the PDB file will also help you.

1. Start with a pdb file read into Insight.
2. You can choose to display certain subsets of atoms under the molecule-display menu. (or use the ON/OFF icon) Under this menu you 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 into the molecule spec box using the formats outlined above.

   Spend about 15 minutes trying all the different options to display various sets of atoms.

3. Molecule-Color
   Explore using this menu to color different parts of the molecule. Note the different methods of specifying the color. Under color method-specification, if you click in the color box, a pallet of colors comes up and you can select one. Coloring by charge/temp factor will be covered in a later tutorial.

Part 4: Molecule Renderings*

1. Start InsightII, read a PDB file into the program.
2. On pchem01-pchem06 (the workstations in room A202), Click on Session, Environment, and RGB_Mode, and click on the blue box next to "RGBMode" at the bottom of this menu to tun RGB mode ON.
3. Molecule-render
   Spend some time experimenting with the different options. i.e. CPK, ball and stick, stick, and lines. Note that CPK rendering take a long time. Therefore you should only try this with a small fragment of your molecule (e.g one or two residues).
4. Go back to the line representation of the whole molecule and cancel the render menu.
5. Molecule-ribbon
   Spend a few minute exploring the different types of ribbon representations. Note that the plane of the ribbon is the plane of the peptide bonds.
6. Molecule-color ribbon
   Experiment with coloring the ribbon in different ways.
7. Delete the ribbon and go back to the line representation of the whole molecule.
8. Molecule-Secondary render: Experiment with different secondary structure representations
   Session-light source: try highlighting different features by changing the apparent direction of the light shining on the molecule.
9. Molecule-surface
   Experiment with different surface representations. Surfaces are fairly slow to calculate, so you might want to choose just a subset of residues for this. The program lets you proceed while the surface is being calculated - this can be confusing.
   Molecule-color: Choose surface and color the surface.
10. On pchem01-pchem06 (the workstations in room A202), Click on Session, Environment, and RGB_Mode, and click on the yellow box next to "RGBMode" at the bottom of this menu to tun RGB mode OFF. Repeat some of these display options (CPK, ribbon, etc.) with RGB mode off.

Part 5: Useful Session and Environment Options

1. Start with a pdb file read in to Insight and rendered/colored to your liking (do not use a surface or CPK representation as this will slow you down too much).
2. Session-help: Experiment with getting help information on various commands
3. Session-autosave: Allows your session to be automatically saved at regular time intervals. The session is saved to a file called ".insight_session.psv".
   SAFETY TIP: In a UNIX shell, you must type "ls -a" to list files that start with a period (".")
4. Session-history
   Lists the commands you have given during this session. Commands are listed in the UNIX shell from which you opened InsightII
5. Session-stereo
   Allows you to display the molecule in stereo - for 3D viewing If you choose the HARDWARE stereo mode, you need special glasses, available only on chemvgx and in room A400 (room A400 is also known as the StereoView room), to be able to see the image in 3D. If you do NOT choose the hardware stereo mode, you will see a "stereo pair" (two images side-by-side that look like 3D images if you cross your eyes or get a pair of stereo lenses; stereo pairs are often seen in publications).
6. Session-Change_directory
   Allows you to change the UNIX directory in which you are operating InsightII
7. Session-Environment-Background
   Allows you to choose the color of the background - black and white are most common
8. Session-Environment-RGB mode
   Turns on RGB mode to allow higher quality images of CPK renderings The disdvantage of using this is that it is somewhat slow. Try it for a CPK representation of a small part of your molecule

Part 6: Saving and Restoring Folders

The represntation is saved in a UNIX file (in the UNIX directory that you specify). The file has the suffix .psv and is refered to by Insight as a "folder". This folder contains all the information about the atomic coordinates, display specifications, renderings, colorings etc. that you have specified using InsightII

• A .psv folder is NOT A DIRECTORY
• A .psv folder is NOT A POSTSCRIPT (.ps) file - IF YOU TRY SENDING THIS FILE TO THE PRINTER YOU WILL WASTE A HUGE AMOUNT OF PAPER!!!
• A .psv folder is NOT A TEXT FILE - you cannot read the contents of this file except by recalling it into InsightII.

1. Start this section by calling a structure up and making a represntation that you like. You might use ribbons, coloring, surfaces, a small section of CPK rendering etc.
2. File-Save_Folder
   Give the name of the object you want to save (typically just * if you want to save everything). Select the directory and type the folder name. Execute.
3. Go to the appropriate directory in a UNIX shell Type ls -l to check that the file is there and see how big it is.
4. Back in InsightII, type delete *
5. File-Restore_Folder
   Select the folder and wait for it to be restored.

Part 7: Annotations and Objects

1. Again, start with a molecular representation of your choice.
2. Object-List
   In the parameters window, click on assemblies or objects to get a list of the assemblies or objects you currently are working with. At this stage you probably only have one object, the molecule you are viewing.
3. 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". This is the assembly (group of objects) that the new annotation will be associated with. 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.
   • Click the left mouse button when the cursor is positioned where you want the annotation to appear.
   • Or 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.
4. Object-List
   In the parameters window, click on assemblies or objects to get a list of the assemblies or objects you currently are working with. Now there is an additional object called "ANN" which is your first annotation. It is part of an assembly called "ANNOTATION".

   The various other commands under the Object menu will allow you to copy, paste, rename, delete, blink an object.

5. Transform-Connect
   Select World and execute. This allows you to connect simultaneously to all objects. Try using the mouse to move objects- they should all move together.
6. Transform-Connect
   Experiment with choosing individual objects or assemblies and try using the mouse to move them. This should give you a feel for the usefulness of defining annotations as separate objects. We will learn about some other types of objects later.
7. User-Annotate
   Now experiment with the various types of annotation that you can add and how they can be defined as objects/assemblies and moved by connecting to them.

Part 8: Measuring Lengths and Angles

1. Start with a simple stick representation of a molecule
2. Measure-Distance
   First try determining a distance manually. Click on add. Click on the first atom. Click on the second atom. Note that the distance now appears on the screen. Try experimenting with the add, remove and clear options and try varying the minimum and maximum distances and the distance monitor color.
3. Once you have a few distances defined on the screen, click on List_Defined, Execute. Note the list of distances in the UNIX window. Now click on Output_File and type in a filename, Execute. Go to your UNIX shell and have a look at the file you have created.
4. Clear all the defined distances.
5. Now, try defining a list of distances automatically. Click on Add. Specify Atom 1 as a particular atom (either with the cursor or by tyoing the spec). Specify Atom 2 as any atom in the same molecule (e.g. MOL:A*). Specify Min Distance = 10.00. Specify Max Distance = 12.00. Now execute. This should give a list of all atoms in the same molecule that are between 10 and 12 Angstroms from the specified atom. List the defined distances to an output file and look at the results ina UNIX shell.
6. Measure-Angle
   Try manually measuring specific bond angles. Clear selection.
7. Measure-Dihedral
   Try manually measuring specific dihedral angles. Clear selection.
8. Measure-HBond
   Specify the whole molecule under each molecule spec. Execute. It takes a few seconds to search all potential pairs of H-bond donors and acceptors for those within H-bonding distance. It is easier to see the H-bonds displayed if you turn off the sidechains. List H-bonds to a file.

Part 9: Subsets

1. Start with a simple stick representation of a molecule.
2. Let's define a a helix as MY_HELIX:
   Identify the range of residues in an alpha helix in your structure. You can use Molecule-Display-Only-Atoms-Backbone to display only backbone atoms, or use Molecule-Ribbon to make a ribbon drawing of the backbone. Then click on the first residue of the helix; write down the number of the residue that appears at the bottom of the screen. Click on the last residue of the helix; write down the number of the residue that appears at the bottom of the screen.

   Subset-Define
   Type in a subset name (e.g., MY_HELIX). To define the residues of the helix, the collection level should be monomer/residue. (When would you use an "atom" or "molecule" collection level?) Search_Domain = your molecule. property level = monomer/resudue. property = Mol_Spec (Click on Mol_Spec and see the list of other properties which you can use to define a subset. Can you think of occasions when you want to define a subset using other properties?) value range = same as search domain. The box next to "Color_Subset" should be yellow; if it isn't, click on this box. Then click on the box under "Color" and choose your favorite color.

   Display only the subset MY_HELIX. Measure hydrogen bonds only in MY_HELIX.

3. Let's define an active site in your molecule.
   Subset-Zone
   Type in a subset name (e.g., ACTIVE_SITE). Click on the box under "Center of Subset" and click on an atom in your (putative) active site. Set collection level to monomer/residue. Choose a 10 angstrom Radius of Subset. Color the subset. Your ACTIVE_SITE subset contains all residues which have at least one atom within 10 angstroms of your "Center of Subset" atom. What would happen if you set the collectin level to "atom" or "molecule"?
4. Let's make an assembly of two molecules:
   Click on Object-Copy, click the box next to "displace", and copy your molecule to a new name. Do this again to make a third copy (of course, choose a completely different name for the third object), but turn the "displace" option OFF (click on the box next to displace until it turns blue). The third object is there, but it is EXACTLY SUPERIMPOSED on the origional object! Click on object-Delete, and delete the third object.

   Move one molecule relative to the other so that they almost touch, but they do not overlap.

   Click on Assembly-Associate, select the two molecules, and type in a New Assembly Name (e.g., COMPLEX). Click on Transform-connect, and connect & move the assembly.

5. Let's identify the interface between the two molecules.
   Click on Subset-Interface. The Center of Subset is one molecule. The Search Domain is the other molecule. Choose an atom collection level. Choose a Radius of subset (e.g., 8 angstroms). Color the subset. You have just defined all residues in the second molecule that have at least one atom within 8 angstroms of the other molecule. Try to define all ATOMS in the FIRST molecule that are within 12 angstroms of LYS and ARG residues of the SECOND molecule. molecule.