Using Protein Explorer to Visualize the Mpro Protein
In the first part of this tutorial you identified the amino acid sequence of the SARS virus and noticed that there were minor differences between different strains of the virus. Now you are going to use a program, Protein Explorer, that is available in the workbench panel or you can link to the web. The two protein sequence that we are going to use is: 1Q2W = SARS Mpro protein. First we will look at the 3-dimensional view of the protein and then we will highlight the active site.

WARNING: It is important that you follow these directions step by step and do everything exactly as it says in this tutorial. The Protein Explorer software may crash if you try to move through it too quickly. This tutorial includes several warnings to prevent you from moving ahead too quickly¡Kif you pay attention to them, you should not have a problem.

1. Go to the Protein Explorer website in workbench panel. You will arrive at a new screen where you need to type in the name of the protein that you would like to see. Type in ¡§1Q2W¡¨ in the first space provided. This will bring up the Mpro protein for us to see. Once you have typed in ¡§1Q2W¡¨, click on the ¡§Go¡¨ button.

2. Be patient: it may take a few seconds to load the information. On Windows machines an intermediate window may appear ¡V if so, click ¡§Start Explorer Session¡¨. This window will appear. DO NOT press the ¡§OK¡¨ button until the red ¡§Busy¡¨ sign says ¡§Ready¡¨. Once you have pressed the ¡§OK¡¨ button AFTER the sign says ¡§Ready¡¨, we need to do a few more things to make sure that the program does not crash.

3. Click on the ¡§Toggle Spinning¡¨ button. This will stop the molecule from spinning which can cause the computer to crash.

Once you have stopped the spinning, we are going to hide the water molecules because the can prevent us seeing the things we want to see. Click on the button that says ¡§Hide/Show Water¡¨. At this point, if you have followed the directions correctly, the molecule should look like this:

If you do not see this on your screen, re-read the directions and start over.

4. Before we move on, let¡¦s take a look at our Mpro protein. Place you cursor (mouse arrow) on the molecule and hold down the left button on the mouse (or the only button on the mouse if your mouse only have one button). Now move the mouse around. This will allow you to rotate your molecule and look at it from any angle you wish. We will use this technique later on to find the active site of the protein.

5. Now, we are going to find the active site of the protein. In the box on the top left, click on ¡§Explore More!¡¨.

The screen on the left will change. Now, we are going to make some changes to our molecule that will make it easier for us to see what is going on.

6. First, let¡¦s change the way the protein looks. Click on the menu that says ¡§Display¡¨ and highlight ¡§Cartoon¡¨ in the drop-down menu. IF YOU WANT TO PLAY AROUND WITH THE DIFFERENT TYPES, YOU CAN CHOOSE ANY OF THE CHOICES ABOVE THE LINE (SPACEFILL AND ABOVE) TO SEE WHAT THE MOLECULE WILL LOOK LIKE. WE WILL NOT USE ANY OF THE CHOICES BELOW THE LINE. Before you continue, make sure that ¡§Cartoon¡¨ is highlighted before continuing with the tutorial.

7. Now we are going to change the color of the protein. As of now, it appears as two colors because two of the same smaller proteins join to form one protein. This is called a protein dimer. Click on the menu box that says ¡§Color¡¨ and choose ¡§Blue¡¨.

8. Now we are going to get more specific. We know that all proteins have an active site on them. So we are going to change the color of one of the smaller monomers (two monomers make up the dimer) so that it stands out from the rest of the protein. This will make it easier for us to see where the active site is. Click on the ¡§Select¡¨ menu and choose ¡§Chain A¡¨.

Nothing visible should happen right away after you choose this. Go to the ¡§Color¡¨ menu and choose the color ¡§Red¡¨. After you have chosen ¡§Red¡¨, Chain A in your protein should turn red. Rotate the protein with your mouse again to see what the protein looks like now. As you can see, the red chain and the blue chain are actually identical to each other. They are stuck to each other in a diagonal way.

9. If you have done everything correctly so far, your protein should look like this:

Now that you can tell that there are really two proteins making up the SARS protein, let¡¦s take a closer look. Because amino acids make up proteins, the active site of this protein should be made of amino acids also. Let¡¦s see if we can find it

10. Click on the link that says ¡§Advanced Explorer¡¨ (you may need to scroll down)
REMEMBER: DO NOT PRESS ANY KEYS OR BUTTONS WHEN THE RED ¡§BUSY¡¨ SIGN IS ON THE SCREEN UNDERNEATH THE PROTEIN! IF YOU PRESS SOMETHING, THE PROGRAM MIGHT CRASH!

11. After the ¡§Busy¡¨ sign has changed to ¡§Ready¡¨, click on the link that says ¡§Seq3D¡¨. This is going to allow us to view individual amino acids on the protein. We will use this tool to find out where the active site on the protein is.

12. Go to the ¡§CPK Colors¡¨ menu and choose ¡§Sec¡¦y Structure¡¨. Nothing should have happened to the protein yet. This is going to allow us to pick specific amino acids to highlight.

13. Now, look at the bottom left-hand panel. Each one of those letters represents an amino acid in the protein sequence. We want to view the amino acids that make up the active site of the protein.

Each of the 20 amino acids has a specific letter that represents it (F-phenylalanine, R-arginine, K-lysine, etc.). As you roll the cursor over a letter, the text box above the sequences tells you what amino acid you have the cursor on. It also tells you the position of the amino acid (the first F in the A chain represents phenylalanine and it is in the number 3 position). We can use this information to find the active site.

The sequence of the active site of this protein is:

The positions of the amino acids are indicated under the sequence. The first amino acid of the active site is cysteine (C) at position 22. Glycine (G) is next at position 23. With your mouse, click on the ¡§C¡¨ at position 22 on chain A. When you click on the C, the site at which this amino acid is at should be visible on the protein. It should appear as a colored bump. Next, click on the ¡§G¡¨ at position 23. Then, click on the ¡§T¡¨ at position 24 and 25. Continue to click on the amino acids that correspond to the active site sequence. When you are done you should be able to see the active site and your protein should look like this: