Molecular Visualization

Dr. Lorraine Marsh

RasMol is a freely available program to visualize and manipulate biological molecules. We will use it to examine DNA, Protein and Protein-DNA complexes.

Click on the rasmol icon (three linked balls).

From ‘file’ menu choose ‘open’ and open ‘myFile.pdb’ (substitute file you want) by clicking.

Image should display. Click and drag to rotate.

Experiment with options in ‘view’ ‘color’ and ‘options’. Some combinations may be better than others. E.g. try ‘view – spacefill’, ‘color – chains’, ‘options – specular, shadows’. Do not print images, but if you want to save, you can choose ‘export’ menu and save as a GIF file (e-mail to a friend?) If image gets messed up by your changes, it is usually fastest to just use ‘file’ close and then open the file again to get a fresh image to work with.

Question: can you read the sequence of this DNA? (Hint: use ‘options’ labels).

For more options there are several HTML-based manuals you can read in the RasMol folder.

Open double-stranded DNA.pdb file. Identify the major and minor grooves of DNA, ribose phosphate backbone and the base-pairing interactions. Blow up a region for close viewing. Change coloring options to see various features better. To what type of atom in A,T,G, and C does the deoxyribose connect? Identify phosphate groups. Can you distinguish 5’ and 3’ oxygens on the deoxyribose ring? Change the display to spacefill which is closer to the real appearance of DNA. Is there access to the bases via the major groove? The minor?

We will study how a mutation causes its effect. Go to ‘file’ menu and close the DNA file. Now using ‘file’ open 2HBS.pdb, deoxyhemoglobin S. The image, in wire-frame view opens. The hemoglobin PDB file has about eight hemoglobin monomers in it. We need to focus on one molecule. To do this we use the RasMol command screen. Click on the RasMol command button on the tool bar at the bottom of the screen if it is reduced. You get a new text window. Type:

after hitting return the last time, click on the image screen. You should now see only one hemoglobin monomer (monomer ‘a’) colored white, with the heme iron-binding group (heteroatoms, not protein) colored red. Click on a residue on the image and go back to the command screen by clicking on it. The residue number should be reported.

Question: can you find Val6 (valine residue 6)? Note that we are looking at HbS the sickle-cell anemia version of hemoglobin. In the wt hemoglobin A residue 6 is glutamate.

Experiment with different views of hemoglobin. Can you see the heme groups with the spacefill view activated? Can you see residue 6? Is residue 6 on the surface of the protein? The sickle-cell version of hemoglobin interacts with itself. Can you explain why this might be?

Using the ‘file’ menu, close hemoglobin.

Open the bacterial lac repressor DNA binding protein (lac.pdb) bound to DNA.

From ‘view’ menu choose backbone.

Can you distinguish DNA and protein? (There are only a few bases of DNA in this file)

On the command window type carefully:

Now you should see the DNA protein complex with the protein residues touching DNA highlighted. Where does the regulatory protein touch the DNA? Click on a residue of polymerase that touches DNA. In the command window the name of this residue (e.g. Met45) will be listed. Record at least two residues that touch DNA.

What part of DNA polymerase serves as a clamp?

Now type on command window:

Close the file and reopen it to clear formatting. Experiment with commands like:

Question: What residues of DNA make contact with the lac repressor? (Try using the ‘select within’ command modified to highlight DNA).

Repeat with the estrogen receptor (1HCQ.pdb). How do the bacterial and mammalian regulators differ?

Open the nucleosome file (1AOI.pdb). Use ‘select protein’ and ‘select dna’ as above to distinguish the DNA and histone components of this large complex. Experiment with display options (you may want DNA in wireframe and protein in spacefill). Highlight each subunit by using

etc. etc.

How many histone subunits are present? What residues in histones (the histones are all similar, but not identical) are touching DNA? Some residues of histone stick out from the complex and are targets for modification that controls DNA structure (e.g. in X inactivation). Are the residues of histone that stick out located at the beginning or end of the molecules? (Click on extended residues in the RasMol window and their residue number will be displayed in the command window. Low numbers like 1-20 are at the beginning of the molecule and high numbers, like 300-400 are near the end.)