Name: _________________

1. Login to the workstation as "C581" using the password discussed in class (note that UNIX is case-sensitive).
2. Open a UNIX shell and change to your directory (cd lastname_firstname).
3. Start Insight by typing "insight2" in the UNIX shell.

1. Go to "File" and choose "Restore_Folder". Select the file "B_DNA_duplex.psv" from the list of files in the right window. Click on Execute and wait for the molecule to appear. Click on Cancel to remove the dialog box.
2. You can color the molecule (using the "colat" command) to get a better view.
3. (a) Rotate the DNA so that a G:C base pair is visible. Look for the possible Watson-Crick hydrogen bonding interactions. Note: Clicking on an atom will tell you the type and number of the atom (e.g. N1).

Measure the lengths of hydrogen bonds in this G:C base pair. To do this, click on "Measure"-"HBond". Choose ‘B_DNA_1’ and ‘B_DNA_2’ for the two "Molecule Spec" boxes, respectively. Clicking on Execute will display all the H-bonds. Finally, measure the angle between any three atoms in a 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.

 

 

 

 

 

 

 

 

 

 

(b) 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.

 

(c) 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.

 

 

 

 

 

 

 

 

 

 

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 –

2’ hydrogen (from the deoxyribose ring) –

 

 

4. Label the major and minor grooves. To do this, click on "User" and select "Annotate." Select "Text". Under "Attach to" it should say "annotation". Type the desired text in the "Text" box. Click in the "X-Coord1" box, and then move the cursor off the window and over to the protein. Either (a) click the left mouse button when the cursor is positioned where you want the annotation to appear, or (b) hold down the left mouse button; 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 again.

 

5. Save this file in your directory by selecting "File" and then "Save_Folder." Click in the "Folder Name" box and type a new name for the file. Click on "Execute."

 

6. It may be easier to delete the labels from the figure before proceeding. To do this, click on "Object" and select "Delete". Then to delete all the annotations select all the ones which start with "ANN", one by one. Do NOT delete the actual molecule.

 

7. D_z, the vertical displacement per base pair, is also called the "rise". Think of a way of measuring the displacement per base pair in a particular DNA strand. Record the value below.

 

 

 

8. 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." For this you will choose two atoms in the plane of the base and then two atoms in the plane of the sugar. The convention for this angle is to choose O4’-C1’-N1-C2 for pyrimidines and O4’-C1’-N9-C4 for purines. The dihedral angle should be almost the same for any of the bases in B-form DNA. Record this value below. Is this the syn or anti conformation?

 

 

 

9. 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.

 

 

10. Look at the base pairs and observe how each base in that pair interacts. 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?

 

 

11. Delete this molecule.

"Restore Folder" named "A_DNA_duplex.psv".

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. Try to compare and contrast A- and B-form DNA based on their structures. For example, comparison of the major and minor grooves, overall length & breadth, base stacking etc.

 

3. View the helix down the helical axis. Do you notice any difference from the B-DNA?

 

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

N7 of purines –

N3 of purines –

2’ hydrogen (from the deoxyribose ring) –

 

Are they any different from B-DNA?

 

 

 

5. Measure the rise in A-DNA and record the value below. How is it different from the rise for 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 this value different from that for B-DNA and what is its major consequence to the overall structure?

"Restore Folder" named "Z_DNA_duplex.psv". Note that this only has the G:C and C:G base pairs.

1. Measure the dihedral angle for both a G and a 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 the values for B-DNA and A-DNA?

 

 

 

3. Delete this molecule.

1. "Session"-"Quit", then Execute will allow you to exit from "Insight". List the files in your directory by typing "ls".
2. "Exit" all windows and "Log out".