Names _________________

Useful UNIX and Insight Questions

1. Login to the workstation as "C581" using the password supplied 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. McClain_Diana) by typing "mkdir lastname_firstname". Change to your directory by typing " cd lastname_firstname". Note: both people in your group should make their own directory because you will need them for later assignments. Any work you do on these computers must be done under either your own or your partners directory.

5. Start the modeling program by typing "insight2". This will take a couple of minutes for the program to load.

6. 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.

For the remainder of this problem set, it will be helpful to have the book and any class notes in front of you.

1. Go to "Molecule" and choose "Get".

Choose "PDB" in the Get File Type box and "System" in the PDB Directory Box. Open the file titled 1d16.pdb. This file name should appear in the file name box. Click on "Execute" and wait for the molecule to appear.

Click on "Cancel" to remove the dialog box.

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

   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.

   The various mouse buttons do the following:

   • 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

The part of the molecule that is observed is determined by the thickness along the z-axis. This is easiest to visualize from a side view. Click on the icon containing a face profile to get a side view.

1. Type "colat" at the Command line. This colors the atoms according to atom type: blue = nitrogen; red = oxygen; green = carbon; white = hydrogen; yellow = sulfur; pink = phosphorus

2. Type "delete *" at the Command line to delete the entire molecule from Insight’s memory.

    

Now, you are going to build you own B-DNA helix.

Click on the blue and white star-like icon in the upper left corner.

Select the "Biopolymer" option. A second row of commands should appear at the top of the screen.

Choose "Nucleotide" and then "Append."

Make sure the B_DNA_Duplex is selected.

Name your molecule in the "Molecule Name" box.

Next, click on the "Nucleotide" box. This will allow you to choose from the 4 base pairs.

Pick one of these, allow it to build, and repeat 10-12 times using any combination. This should allow you to observe at least one full turn.

If it is easier to view the molecule, you can color the molecule using the "colat" command.

1.  
   1. Rotate the DNA so that a G:C base pair is visible. Look for the possible Watson-Crick hydrogen bonding interactions. Draw a G:C base pair below. You can either use your book or the DNA model to draw the base pairs. Note: clicking on an atom will tell you the type and number of the atom (e.g. N1). Measure the distance of one hydrogen bond and the heavy atom distance. To do this, click on "Measure" and then choose "Distance." Then, select the two atoms to measure the distance between. How do your measured distances compare to those found for strong hydrogen bonds? Finally, measure the angle between the three atoms in the hydrogen bond. To do this, click on "Measure" and then choose "Angle." Next, select the three atoms in the angle. How does your value compare to what is expected?

      Do the same for an A:T base pair.

   2. Rotate the DNA so that you are viewing it down the helical axis. Notice that the phosphate groups are on the outside of the helix and the bases fill the center. Sketch this below.

   3. Rotate the DNA and look at the sugar and phosphate groups. Identify the 5’- and 3’ carbons. Sketch a ribose ring and the attached phosphate. Number the ring.

2. It will be important for you to be able to identify the major and minor grooves. Observe that one groove is wide and deep and the other is narrow and shallow.

   Look at the base pairs in the molecule and label the following atoms as being in the major or minor groove:

   N7 of purines –

   N3 of purines –

   O2 of pyrimidines –

   N2 of guanosine –

   O4 of thymidine –

   2’ hydrogen (from the deoxyribose ring) –

   4’ hydrogen –

   Label the major and minor groove. To do this, click on "User" and select "Annotate." Select "Text" and then click in the "Attach to" box. Select the name of your molecule. Type the desired text in the "Text" box. By selecting the x-coordinate and holding down the left mouse button, the text can be dragged to anywhere on the screen. Draw an arrow to the N7 and N3 of a centrally located purine.

   Save this file in your directory by selecting "File" and then "Save_Folder." Click in the "Folder Name" box and type the name of the file. Click on "Execute." Indicate below whose directory the file is saved in and the name of the file.

   It may be easier to delete the labels from the figure before proceeding. To do this, click on "User" and select "Annotate." Choose "Undo" until all the labels have been removed.

3. D_z, the vertical displacement per base pair, is also called the rise. Determine which is the 5’ end and which is the 3’ end of a single DNA strand. Measure the rise by clicking on "Nucleotide" and selecting "Measure. " Click on the 5’ end for the 5’ boxes and the 3’ end for the 3’ boxes. You will click on each end of the molecule twice. To find the rise, click on "Texport On" at the bottom of the screen to view the UNIX shell. Scroll up to find the displacement value, which is the same as the rise. What is this value? Click on "Texport Off" to remove the UNIX shell.

4. The angle between the sugar and the base will also affect the overall shape of the molecule. Measure the dihedral angle for any one of the bases by selecting "Measure" and then choosing "Dihedral." The dihedral angle should be the same for any of the bases in B-form DNA. First, you will choose two atoms in the first plane and then two atoms in the second plane. The convention for this angle is to choose O4’-C1’-N1-C2 for pyrimidines and O4’-C1’-N9-C4 for purines. Record the value below. Is this the syn or anti conformation?

5. Now, let’s look at the sugar pucker. The easiest way to view this is to line up C1’-O4’-C4’ in a plane and observe the twist of C2’ and C3’ relative to that plane. What type of pucker is observed? Measure the distance between the 5’ and 3’ phosphorus atoms attached to the same sugar.

6. Look at the base pairs and observe how the individual bases in that pair interact.

   Choose "Object" and select "Axes_Display." Notice that the two bases in a base pair are not exactly in the same plane. What is this property called?

   Why are the bases tilted?

   Delete this molecule.

    

Make a new helix with A_DNA_Duplex.

1. The terms major and minor groove are based on the B-DNA structure. Because of the shape of A-DNA the grooves are sometimes referred to as narrow and wide. Which groove (major or minor) would be classified as narrow in A-DNA? Which groove (major or minor) would be classified as wide?

2. Without using your book, compare and contrast A- and B-form DNA based on their structures. Be sure to include a description of the major and minor grooves.

3. Are the following atoms in the major or minor groove:

N7 of purines –

N3 of purines –

2’ hydrogen (from the deoxyribose ring) –

4’ hydrogen –

Is there any difference from B-DNA?

4. Measure the rise in A-DNA and record the value below.
5. Measure the dihedral angle for any base and record the value below. Is this the syn or anti conformation? Compare this value with the one obtained for B-DNA and describe the orientation of the base relative to the sugar for both A-DNA and B-DNA.

6. Finally, look at the sugar pucker in A-DNA. The easiest way to observe this is to line up C1’-O4’-C4’ in a plane and observe the twist of C2’ and C3’ relative to the plane. What type of pucker do you observe? Measure the distance between the 5’ and 3’ phosphate atoms and record the value below. How is that value differ from B-DNA and what is the major consequence to the overall structure?

    

Finally, we will look at similar parameters for Z-DNA. Make a Z DNA Duplex. You are only given the option of a G:C or C:G. Alternate these two to get a typical Z-DNA.

1. Measure the dihedral angle for both a G and C base. Are they in the syn or anti conformation? If it is in the syn conformation, which atom is over the ring?

2. Find a guanosine. What type of sugar pucker do you observe? Once again, measure the distance between the 5’ and 3’ phosphate atoms. How does this compare to B-DNA and A-DNA?

3. Finally, briefly compare and contrast the three forms of DNA that you looked at today.