C687: Introduction to InsightII

Parts 0 to 5 constitute the first part of this tutorial.

Part 0: Preparing BEFORE the tutorial session

Part 1: Getting Started

1. In a UNIX shell, cd to the appropriate directory.
2. Type insight2 or insightII
3. Wait for InsightII to start up.
4. Take a few minutes to familiarize yourself with the layout of the InsightII 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 and identify various options that you learned about from Part 0 of this tutorial.
   • 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. InsightII is case-insentive: DELETE MOLECULE_1 and delete molecule_1 are the same command. All typing in other InsightII menus and windows is also case-insensitive. The only exception is when you specify UNIX files and directories, which ARE case-sensitive.
   • Panel in lower left corner to facilitate molecule rotations/translations etc.
   • Option keys along bottom to perform various useful commands. In particular, click on Textport On to bring the textport to the forefront, and click on Textport Off to put the textport behind the InsightII window. DO NOT iconize the textport window (see next step).
5. Click on the "dot in a square" on the upper right hand edge of the InsightII 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 can be very helpful.
7. DO NOT double-click on the box in the upper left corner of the window. Double-clicking on this box will EXIT the window system, killing the program. DO NOT single-click on thix box with the right mouse button and select exit. DO NOT double-click or select exit from the box in the upper left corner of any other InsightII window (menu window, textport window, etc.), or you will kill the entire program.
8. Go to Session/Quit to exit from InsightII. Confirm exit. It takes a minute for the program to shut down.

Part 2: Getting and Moving Molecules

1. Download the zinc finger PDB file from this web site.
2. Start up InsightII again.
3. Select the Molecule/get menu.
   Click on the button next to PDB to display only files that end with .pdb Click on the name of your zinc finger PDB file.
   If you desire, change the molecule name under Get Molecule to a name that you prefer.
   Execute and wait for molecule to appear.
   
4. Select the Molecule/put menu.
   Choose the pdb file format.
   In the file name box, type a name for the new file.
   Click on Execute
   Do a directory listing in a UNIX shell:
   • open a new UNIX shell via the desktop toolbox. cd to the appropriate directory, and type ls to display the directory list.
   • in InsightII, type unix, hit the <enter> key TWICE, and the textport will pop forward and the UNIX command prompt will appear. Type ls to display the directory list. Type exit to return to InsightII.
5. Back in InsightII, spend some time exploring the ways that 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. See also Mouse Movement Options.

   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.

   Explore using the panels in the lower left corner to effect molecular rotation, translation, world scale, and slab thickness. These are described in the Dial Slider Box section.

6. Type on the command line delete *. This will delete all objects you currently have in InsightII.

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 strand A in the pdb file containing the IL8 structure
IL8:B*	specifies all atoms in strand B in the pdb file containing the IL8 structure
IL8:A25	specifies all atoms in residue number 25 in strand A
IL8:A25-A32	specifies all atoms in residues 25 to 32 in strand A
IL8:A25,A30,A35	specifies all atoms in residues 25, 30, and 35 of strand A
IL8:Cys	specifies all atoms in all Cys residues in either strand A or B
IL8:B50:N	specifies the amide nitrogen in residue 50 of strand B
IL8:B50:CA	specifies the alpha carbon in residue 50 of strand 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 and click (with the left mouse button) 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 in InsightII's black background area to deselect. The atom and molecule labels in the PDB file will also help you.

1. Start with a pdb file read into InsightII.
   If you don't have a PDB file, first download the zinc finger PDB file from this web site.
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 displayed. 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 10 minutes testing different options to display various sets of atoms.
3. Select the Molecule/Color menu.
   Using this menu for ~10 minutes to explore how to color different parts of the molecule. Note the different methods of specifying the color. If you click in the color box, a pallet of colors pops forward and you can select one of the pre-defined colors, or you can use the slide bars to create a new color. Coloring by charge or temperature factor will be covered in a later tutorial.

Part 4: Molecule Renderings

1. Start InsightII, read a PDB file into the program.
   If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. Select the Molecule/Render menu.
   Spend some time experimenting with the different options. i.e. CPK, ball and stick, stick, and lines. Note that CPK rendering can take a long time. Therefore you should only try this with a small fragment of your molecule (e.g one or two residues).
3. Go back to the line representation of the whole molecule and cancel the render menu.
4. Select the Molecule/Ribbon menu.
   Spend a few minutes exploring the different types of ribbon representations. Note that the plane of the ribbon is the plane of the peptide bonds.
5. Select the Molecule/color ribbon menu.
   Experiment with coloring the ribbon in different ways.
6. Delete the ribbon (using the Molecule/ribbon menu) and go back to the line representation of the whole molecule.
7. Select the Molecule/Secondary render menu.
   Experiment with different secondary structure representations.
   The PDB secondary structure definitions may not be present, so only the Kabsch & Sander definitions can be used.
8. Select the Molecule/Surface menu.
   Experiment with different surface representations. Surfaces are fairly slow to calculate, so you might want to choose just a subset of residues for this. Also, choose a LOW surface resolution to make this calculation faster. The program lets you proceed while the surface is being calculated - this can be confusing.
   
9. Select the Molecule/Color menu, choose surface and color the surface.
10. Delete all renderings.

Part 5: 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 called an "InsightII 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. Get a molecule and make a representation that you like. You might use ribbons, coloring, surfaces, a small section of CPK rendering etc. If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. Select the File/Save_Folder menu.
   Type in the name of the object you want to save (typically just * if you want to save everything), or select an object in the Parameter menu. Type in first_folder.psv for the name for the new folder. Execute.
   WARNING: DO NOT use the replace option. ALWAYS type in a new, unique file name. The replace option does NOT work in a logical way: It will save an object in the old folder ONLY if there is already an object with the same name already in the old folder. This can be very confusing, and can lead to lost work if you are not careful, so DON'T USE the replace option.
3. Go to the appropriate directory in a UNIX shell.
   Type ls to verify that the file is there. Type du -sk filename (where filename is the name of your file) to show the size of this file.
4. Back in InsightII, type delete *
5. Select the File/Restore_Folder menu.
   Select the folder and wait for it to be restored.
6. Type delete * again.

1. Start with a pdb file read in to InsightII and rendered/colored to your liking (do not use a surface or CPK representation as this will slow you down too much). If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. Select the Session/help menu.
   Experiment with getting help information on various commands.
3. Select the Session/autosave menu.
   This feature 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. Select the Session/history menu.
   This menu lists the commands you have given during this session. Commands are listed in the UNIX shell from which you opened InsightII.
5. Select the Session/stereo menu.
   This menu allows you to display the molecule in stereo - for 3D viewing.
   If you choose the HARDWARE stereo mode, you need special LCD glasses, available only on splatter, stereo3, and in room A400 (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. Select the Session/Change_directory menu.
   This menu allows you to change the UNIX directory in which you are operating InsightII.
7. Select the Session/Environment/Background menu.
   This menu allows you to choose the color of the background - black and white are most common, but dark blue works very well for print-outs.

Part 7: Annotations and Objects

1. Again, start with a molecular representation of your choice.
   If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. Select the Object/List menu.
   In the parameters window, click on Assembly or Object 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. Select the User/Annotate menu.
   This menu allows you to add several different types of annotations. We will use text annotation as an example. Choose text.
   annotation should be listed in the Attach To box. 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 near 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 Undo, and try it again.
4. Select the Object/List menu.
   In the parameters window, click on assembly or object 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. Select the Transform/Connect menu.
   Select World and execute. This allows you to connect simultaneously to all objects. Use the mouse to move objects- they should all move together. You can also click on the Connect World button at the bottom of the InsightII window.
6. Select the Transform/Connect menu.
   Experiment with choosing individual objects or assemblies and use 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. You can also click on the Connect Object button at the bottom of the InsightII window, and then click on an object.

   CAUTION: Molecular objects are 3D---they can move and rotate in three dimensions. Most annotations are only 2D---they can only move and rotate in the X and Y dimensions. Therefore, if you rotate molecules and annotations, their relative placements may not be what you want. It's best to position your molecule(s) in the orientation that you want and then add all annotations WITHOUT readjusting orientations of yoru molecule(s).

7. Select the User/Annotate menu.
   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.
8. Save your best, final rendition of models & annotations in a folder named annotations.psv in your home directory.

Part 8: Measuring Lengths and Angles

1. Start with a simple stick representation of a molecule.
   If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. Select the Measure/Distance menu, and determine 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. Experiment with the add, remove and clear options; vary 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 and Execute. Note the list of distances in the Textport window. Now click on Output_File and type in a filename, then Execute. Go to your UNIX shell and view the contents of the file that you have just created.
4. Clear all the defined distances.
5. Define 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* for the A strand of the molecule MO, or ZINC_FINGER for any atom in the zinc finger moleculeL).
   Specify Min Distance = 10.00.
   Specify Max Distance = 12.00.
   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 in a UNIX shell.
6. Select the Measure/Angle menu.
   Manually measure specific bond angles.
   
7. Select the Measure/Dihedral menu.
   Manually measure specific dihedral angles.
8. Save your model, with angle & dihedral angle measurements, in a file named angle_meas.psv in your home directory.
9. Clear all Angle and Dihedral Angle measurements.
10. Select the Measure/HBond menu.
    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.
   If you don't have a PDB file, first download the zinc finger PDB file from this web site.
2. 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, or Molecule/Secondary Render to render the helices as cylinders. Then close all menus and 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.
3. Select the Subset/Define menu.
   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?)
   Set Search Domain to your molecule.
   Set property level to monomer/resudue.
   Set property to 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?)
   Set value range to the same value 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. Molecule/Display only the subset MY_HELIX. Measure/H bonds only in MY_HELIX.
4. Define an active site in your molecule using the Subset/Zone menu.
   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 collection level to "atom" or "molecule"?
5. 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 original 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.
6. Color all molecules blue.
7. 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 yellow.
   You have just defined all residues in the second molecule that have at least one atom within 8 angstroms of the other molecule. What would happen if you set the collection level to "atom" or "molecule"?
8. Save the models in a file named interface.psv in your home directory.

Part 10: Verify that you have completed this portion of the assignment

See the Viewer Assignment page for details.