Project 3 – Exploring the PDB, Protein Explorer and Molecular Visualization

Instructions

After completing these simple exercises, you should be familiar with how to use many of the basic commands in Chime and Protein Explorer (PE). These programs will allow you to manipulate protein and nucleic acid structures to suit your needs. Chime is a plug-in module that provides extra functionality to web browsers. Many of the web pages we will be using incorporate Chime images. These images can be identified by the small MDL icon in the bottom of the frame and can be manipulated using the mouse. Protein Explorer is a more general program available through the PDB as a viewing program. It uses Chime, but also has a command line that allows you to type in more exact instructions for enhanced viewing and manipulations. You can download Chime onto your computer (Download Chime Now). Follow the directions to install the program. The tutorial below will teach you to use PE. This knowledge will let you manipulate Chime images on other web pages in the same fashion.

One note on color schemes. The CPK color scheme is commonly used in these exercises. In this convention, red = oxygen, blue = nitrogen, gray = carbon, white = hydrogen, and yellow = sulfur.

Follow these steps to access the Protein Data Bank.

1. Open Netscape (Do NOT use Internet Explorer). Use of version 4.7x is highly recommended. Netscape 6.x has some problems with the way it supports Java and PE does not run consistently on 6.x.

There is now a version of protein explorer that will run in Internet Explorer. If you are a die hard IE users, go to:
http://proteinexplorer.org

Once you are at that URL,skip to step 3 below and enter 1IGD in the PDB identification code window in the center of your screen. You will then jump ahead to step 8.

2. Go to http://www.rcsb.org/pdb/index.html. This URL is the Protein Data Bank (PDB) web page. A link is available on the course links web page.

3. Use the Search tool to find entry 1IGD.

4. A summary information window with a sidebar should appear.

5. Click on the View Structure button on the sidebar. Another window should pop up with a list of ways you can view the structure. Choose Protein Explorer.

6. Another window will appear from which you can start Protein Explorer. Follow the instructions on the next few pages until the structure appears (click on the button at the bottom of the page after setting the window size as you would like it. Use the Protein Explorer 2 beta version.)

7. You will have another startup window (this occurs due to the beta version we are using). Click on the Advanced option box and then START SESSION. Wait for the structure to appear and the green ready light to appear at the bottom of the left window.

8. Your window will have 3 frames

• The Structure Window
• First View Window
• Message Window

9. Stop the molecule from spinning by pressing the toggle spinning button at the top right.

10. The large red balls are water molecules. You can remove them by pressing the hide/show waters button.

11. Press the link called Explore More with Quick Views.

12. There are several ways that you can adjust what you are looking at and the instructions below will walk you through some of the manipulations. Anytime you want to reset the image, press the RESET VIEW button and the original file will reappear on your screen.

13. Let’s see what it is we are looking at. Press the Mol Info button. A new window will appear with links to information about the protein you are looking at. Press the link to the sequence. Here you can look at the primary structure of this small protein. Clicking on the boxes and pressing the apply will show you where in the sequence the given amino acids are located. Close this window when you are finished exploring.

14. Now, let’s learn to rotate, zoom and translate the molecule in the structure window.

	MAC	PC
ZOOM	Hold down the SHIFT key and the mouse button drag the mouse back and forth	Hold down the SHIFT key and the left mouse button drag the mouse back and forth
ROTATE	Hold down mouse button move the mouse in a circular motion	Hold down the left mouse button move the mouse in a circular motion
TRANSLATE	Hold down the APPLE key and the mouse drag the mouse up and down or side-to-side	Hold down the CONTROL key and the right mouse button drag the mouse up and down or side-to-side

You can also zoom in and out using the button in the quick view window. Each time you press the button it will zoom an incremental amount.

15. There are 3 general ways manipulate the image. They each have their merits so you will want to become familiar with each of them. These include:

a) Using the quick view menus
b) Typing into the command line (located directly above the box labeled “Recall”) – for this you will need to learn some of the syntax
c) Using the menu located under the MDL icon in the lower right hand corner of the structure window.

16. Let’s start with the MDL icon. Click on it and hold down the mouse button. A scroll bar will appear. You can manipulate the structure using this tool bar.

17. Convert the molecule to spacefilling mode:

MAC	PC
Scroll down the MDL menu to Display. When the submenu opens follow it to spacefill and then van der waals radii. Now let go of the mouse button. The command will act on any part of the molecule selected or if nothing is selected, the whole molecule.	Click on menu, release, and then click on the menu where it says Display. Repeat this process on Spacefill and again on van der waals radii.

This command will be written out as:

(MDL) Display --> spacefill --> van der waals radii

The MDL menu is present in all chime web pages and not just protein explorer. Many of the web pages used in the course include chime structures. This pull down menu is the only way to manipulate structures in the generic chime pages, so you will need to understand basic manipulations if you wish to get the most out of these web sites.

Use this tutorial to get more familiar with the program. Then answer the questions on the problem set by applying what you learned in the tutorial.

1IGD is a small protein with 61 amino acids. It is a domain of a larger protein involved in IgG binding. It has a simple compact structure that will make it a good place to learn to manipulate the images of macromolecules.

The first task is to remove the solvent molecules that make it hard to see the protein.

(MDL) select --> hetero --> solvent
(MDL) select --> hide --> hide selected

The same function can be carried out by using the water button in the upper right window that toggles the waters between visible and hidden. This command would be written as

(Quick View) water

The protein backbone is currently displayed in the spacefilling mode. Leave the waters toggled off. Let's change that to see it in cartoon format.

(Quick View) select --> protein
(Quick View) display --> cartoon

Rotate the molecule and look at the secondary structures present. This would be great view to use to sketch a topology diagram like you did on Problem Set 2.

There are several ways to select particular residues as you can see from the MDL select menu. Let's change the color of the helical region.

(MDL) select --> protein --> helix
(MDL) select --> change color to --> green

You can also use the command line for these manipulations. For instance, let's change the color of the some of the beta-sheet region using the command line. First, we have to select the region of interest and then we have to change the color. These are independent commands and can be mixed and matched at will.

(Command) s sheet --> return
(Command) color red --> return

The selection is retained and you can split the commands between the command line and the MDL menu.

(MDL) display --> sticks

reset the image to cartoon mode.

(MDL) display --> cartoon

When using Protein Explorer, many of the manipulations will be easiest to perform in the Quick Views menus. Reset the view by pressing Reset view and return to the quick view menu after removing the waters and stopping the spinning.

(Quick View) Select --> all
(Quick View) display --> cartoon
(Quick View) select --> helices
(Quick View) color --> yellow
(Quick View) select --> strands
(Quick View) color --> violet
(Quick View) bkg

You will have to select different options to understand just how powerful the program is.

You can also pick out specific residues using the command line

(Command) s 47-51 --> return
(Command) color cpk --> return
(Command) st --> return

You can use the mouse to determine the residue numbers by clicking on the appropriate part of the molecule.

(Command) s 28-42 --> return
(Command) wf --> return
(mouse) click on any atom

The residue number and atom number of the selection will appear in the message window. In a Chime web page where there is no message window, this information will appear at the very bottom of your browser window.

Find residues 29 and 40 in this manner, they are located at either end of the alpha helix, then reset the helix to cartoon mode using the quick views menu.

(Command) s 29 --> return
(Command) color cpk --> return
(MDL) display --> sticks
(Command) s 40 --> return
(Command) color cpk --> return
(MDL) display --> sticks

One thing to note is that there is currently no “UNDO” button. If you are not sure if you have selected the item of interest, simply change its color. That is easy to undo by changing it back to its original state.

You can use the mouse to activate a variety of other functions as well.

(MDL) select --> mouse click action --> toggle atom label
(Mouse) click on one of the side chain oxygens from Glu 29
(Mouse) find on Asn 40 and click on it

If you click on the atom again, the label will dissapear

(MDL) select --> mouse click action --> toggle distance monitor
(Mouse) Find the side chain oxygen of Asn 40 and click on it
oxygen is red in cpk coloring
(Mouse) click on the side chain oxygen from Glu 29
(Quick View) bkg

Orient the molecule so that you can clearly see the distance label and print out a copy to turn in with your problem set.

If your molecule had more than one polypeptide chain, you could use slightly different syntax to pick the correct amino acid(s). The syntax would be:

Command action

(Command) s (1-20:B) select amino acids 1-20 on chain B
(Command) s (*:A) select all of chain A
(Command) s 36,48 select residues 36 and 48
(MDL) select --> chain --> C select chain C

You will have to use the program more to learn all of its nuances. But this quick run through should get you started. If you have specific questions on how to get Chime/Protein Explorer to do something, email your instructor or bring the questions to office hours. Two additional tutorials are available on-line. One is the chime tutorial developed by faculty at CMU, the other is the 1 hour quick tour of Protein Explorer. They take awhile to complete thoroughly but will provide you with more practice using the program. If you need information on how to enter something into the command line, press Show Aliases. A new window will appear with all of the commands and the relevant syntax.

1IGD does not have a substrate bound, but many of the structures of your unknown proteins have ligands (substrates) bound to them. A very powerful command in PE is the show contacts. To take advantage of this feature when exploring the structure of your unknown protein:

(quick view) select ligand
(quick view) display contacts

The resulting image will have the ligand inside an opaque surface and only the parts of the protein within 5 Å of the ligand shown. Everything else will be hidden. Turn off the surface hiding your ligand by

(MDL) select --> display --> toggle visibility

You will then be able to explore how the ligand is bound to your protein with a lot of the other residues removed for greater clarity.

Independent exercises to help you learn about and analyze your unknown protein:

a) Find the structure of your unknown protein. You may already have the PDB record number. If you do not have it, type in the name of your protein in the line labeled “search the archive.” There may be multiple structures. You may pick any of the relevant structures but questions b - d may be easier or harder depending on which one you select.

b) Print out representative views of your protein in as a space filling model. Print out an equivalent view in cartoon mode. Turn in those print outs with this problem set.

c) Use the form "Record of 3D Macromolecular Structure Observations" to guide you as you analyze your protein. If you want to go into even more detail, an advanced form is available ase well.

d) Write a 1-page (typed, double spaced ) description of your protein. Illustrate your discussion with an appropriate figure (made in Protein Explorer) as if it were to be included in a research paper. Click here to read an example of a structure description paragraph. Please see a writing tutor if you have difficulty with this assignment. You will be graded on content and clarity as well as organizational, stylistic and grammatical considerations. (This exercise demonstrates much more than simply that you can use protein explorer. It shows us that you have learned how to look at and think about protein structure.)