1 Hour QuickTour for Protein Explorer
by Eric Martz. Revised July 2, 2001.
Available on-line at molvis.sdsc.edu/protexpl/qtour.htm. Feedback to emartz@microbio.umass.edu.

This QuickTour will introduce you to how to use Protein Explorer (www.proteinexplorer.org) to visualize the major structural features of a macromolecule. It assumes that you are familiar with the fundamentals of protein and nucleic acid structure. If some terms below are unfamiliar, consult an introductory biochemistry textbook.

The QuickTour can be done in one to two hours, depending on how quickly you are comfortable moving ahead. Print this document for handy reference. The purpose of the QuickTour is to organize your introduction to Protein Explorer, not to explain it. Explanations, instructions, and links to further information are built into the pages of Protein Explorer. It is important that you take time to read the information offered within Protein Explorer, because it will not be repeated below in this QuickTour document.

1. If you haven't already started Protein Explorer, start Netscape, and go to www.proteinexplorer.org.
   (Protein Explorer requires Netscape 4, and the Chime 2 chemical structure plugin must be installed. If your browser is not compatible, Protein Explorer will tell you how to make it compatible. It will not work in Internet Explorer or Netscape 6.)

2. First you will see the FrontDoor page. Here, click on the large link Quick-Start Protein Explorer.
   This link loads the DNA-binding domain of the Gal4 transcriptional regulatory protein, bound to DNA. If the molecule does not appear, see the link to Troubleshooting on the FrontDoor. If you are using a Macintosh, try step C5 in the Troubleshooting guide -- it always works!

   In order to show a 3D molecular structure, Protein Explorer requires a data file that specifies the positions of all atoms, called an atomic coordinate file or PDB file. All published macromolecular structures are available from the Protein Data Bank (the PDB). (In the absence of an experimentally determined 3D structure, an amino acid sequence alone cannot be used to see a 3D structure in Protein Explorer. However, sometimes an amino acid sequence can be homology modeled to give an approximate 3D structure.) Each PDB file has a unique 4-character identification code. The code for the Gal4:DNA complex is 1d66.

3. After you start the Protein Explorer session, you should see the molecule, and the FirstView description page. Take some time to digest the information on this page -- it is important!
   You can ignore the slot marked "Commands may be entered here" -- this is for advanced users. Protein Explorer can be used very effectively without entering any commands in this slot.

4. Near the bottom of the FirstView frame is a Molecule Information link. This link will be available at all times (but on later pages will be labeled simply Mol Info). Click it to open the Molecule Information Window, and explore the resources here. If you're in a hurry, the most important one to try is Sequences. (It is best to postpone Seq3D and the other options here until later.)
   The most important things to look for in the Sequences display are gaps and identical chains. (Carbohydrate sequences will not be shown here, but there are no carbohydrates in 1d66.) For 1d66, notice that chain B is identical to chain A. Some PDB files have gaps -- typically regions where the chain could not be resolved. These are easy to spot on the Sequences display as rows of periods "....".

5. Also in the Molecule Information window is an important link to Help, Index & Glossary. Take a look at this. Later on, if you don't know how to get the image you want, or what a term means, look here. This is also where you can find answers to FAQ (frequently asked questions).
   For an example, click on L and read about the Limitations of PE.

6. At the bottom of the FirstView page is a link to a Form for Recording Observations. If you have two or more hours for your QuickTour, you may find it useful to print this and note your observations. If you want to complete your QuickTour in an hour or less, you can skip this form.

7. When you have finished the FirstView page, you should know how many protein chains are in the molecule, how many nucleic acid chains, what ligands are present, whether water is present, and whether any disulfide bonds are present.

8. Click Explore More!, which takes you to the QuickViews menu system. QuickViews is the heart of the user-friendly power of Protein Explorer. Feel free to try anything here that interests you. In the QuickTour steps below, we'll introduce you to the most important capabilities, but by no means all capabilities of QuickViews.

9. Amino and Carboxy Termini. In QuickViews, the DISPLAY and COLOR menu selections always operate on the currently selected atoms. Open the pull-down SELECT menu, and click 'All'. Notice the count of atoms selected below the molecule. Now use the menus to SELECT Chains, COLOR N->C Rainbow. Which end of the protein chain is synthesized first?
   The intact Gal4 protein has 881 residues. Only the 65 N-terminal residues were crystallized in 1d66.

10. Secondary Structure. SELECT Protein, DISPLAY Cartoon, COLOR, Structure. In 1d66, notice the longest two alpha helices that are parallel.

11. Notice that each operation in QuickViews automatically displays detailed help in the middle window. This is where selection terms, display renderings, and color schemes are explained for beginners. It is important that you get familiar with this help each time you do something new -- these explanations will not be repeated here.

12. Try the Top Row of Buttons: Spin, Zoom, Background 'Bkg', Water, and Ligand. These are all toggles (except for Zoom+ and -) so you need to click each one at least twice to see what it does. These buttons are shortcuts to operations that are needed often; they are available on all pages of Protein Explorer (except FirstView).

13. Distribution of Hydrophobic Residues. With Protein selected, DISPLAY Spacefill, COLOR Polarity2. This is easier to see on a black background (press the 'Bkg' button). In 1d66, notice the hydrophobic strip where the two longest alpha helices contact each other. This can be seen most dramatically by using the 'Slab' button. Why are the hydrophobic sidechains gathered in this region?
    Large hydrophobic patches on the surface of a protein (not seen in 1d66) could signal areas of protein-protein contact, or in extreme cases, that the protein sits in a hydrophobic mileu, such as a lipid bilayer. An example of the latter is the potassium channel, 1bl8. If you have more than an hour, at PE's FrontDoor, try the quick-start link labeled Protein Comparator that compares a soluble protein (hemoglobin) with an insoluble one (1bl8).

14. Net Charge. COLOR Polarity5. What do you think is the net charge of the 1d66 protein? How would this support the function of Gal4?

15. Ligand Contacts. Click Reset View (below the cluster of buttons). Click Explore More to return to QuickViews. Now, SELECT Ligand, DISPLAY Contacts. From the menu that appears in the middle frame, choose step by step. Examine the image after clicking each box. You are building an image that is rich in information about the noncovalent bonding contacts to the ligand. After you click the last box, a description of the Contact surface image will appear in the middle frame.
    In the case of 1d66, you will see see the two pairs of cadmium ions, surrounded by the atoms they contact, with the rest of the molecule erased. They will be too small to see clearly -- go to the next step below!

16. Centering. Click the button "Center", then OK in the pop-up question. Click on one of the yellow atoms. It should jump to the center. Click Zoom+ repeatedly until you can see the centered structure clearly.

17. Using Contacts Controls. The ligand (1d66: pair of cadmium ions) is represented by its van der Waals surface. Hide the surface, then click on the "Ligands" button to show the ligand atoms as spheres.
    Hint: If the Contacts help is not visible, click "Restore Contacts Help & Controls". In the Contacts help, scroll down till you see "Surface: Transparent, Hidden, Solid", and click on Hidden. Now click the "Ligand" button until the ligand atoms appear as spacefilling spheres.
    1d66: What element is represented by the yellow balls?
    Hint 1: If the Contacts help is not visible, click "Restore Contacts Help & Controls". In the Contacts help, scroll down till you see the link "balls and sticks are colored by element", and click it. Scroll below the color key and at the bottom of the middle frame, click Back to return to Contacts help.
    Hint 2: Click on a yellow ball and note its identification in the message window (lower left).
    1d66: What element forms a cage around cadmium? How many atoms of this element are bound to the pair of Cd ions?

18. Contacts: Balls vs. Sticks. If the Contacts help is not visible, click "Restore Contacts Help & Controls". Why are some atoms outside the surface shown as balls, while other nearby atoms are shown as sticks? (You can show the surface again by clicking "Surface: Solid" in the Contacts help frame.)

19. Placing Contacts in Context. If the Contacts help is not visible, click "Restore Contacts Help & Controls". Click on "Backbones: Show". This selects all amino acid alpha carbons plus all nucleotide phosphorus atoms (1d66: 150 atoms). Click the "Center" button, and then click Cancel (to center all selected atoms). Zoom to smaller size so you can see where the ligands and their contacts sit in the overall structure.

20. DNA vs. RNA. Click FirstView: Reset View. When the FirstView is restored, click Explore More! to return to QuickViews. SELECT Nucleic. (If zero atoms are selected, you can skip this step.) In the help frame, click the link distinguish DNA from RNA. What do the gray balls represent? Is there any ribose present? (If you have extra time, use this method on 104D.)

When you have time, find a molecule of interest to you, display it in Protein Explorer, and use the above steps to guide your learning about the fundamental structural features of your molecule.

• To find and display your molecule, go to www.pdblite.org. When you have narrowed down your search to one molecule, click the link Protein Explorer in the section "View in 3D".
• If you need a more advanced search tool, check out the resources for finding molecules on the FrontDoor page of Protein Explorer (www.proteinexplorer.org).
• Other ways of loading molecules are listed on the FrontDoor page of Protein Explorer (www.proteinexplorer.org).

This section assumes that you have completed the 1-Hour QuickTour, and are ready to try out more capabilities of Protein Explorer (www.proteinexplorer.org).

In order to restrict it to 1-2 hours, above QuickTour skipped many powerful features of PE. Following the list below will give you an organized overview of most of the important remaining capabilities. The steps below do not offer much explanation, but merely touch upon the capabilities. For more explanation, try the Tutorial (but the Tutorial takes even longer!).

21. Sequence to 3D Mapping. Reset the view of a session on 1d66, or start a new 1d66 session from the first Quick-Start link on the FrontDoor (www.proteinexplorer.org). Open the Molecule Information Window and click Seq3D.
    1. Window control. Seq3D opens a new window with a compact display of the chain sequences. Clicking on, or rotating, the molecule will push the Seq3D window behind PE's main window -- use the Windows Taskbar button marked "Seq3D" to bring it back to the front as needed. (Macintosh: use the Communicator menu to pull Seq3D to the foreground.)
    2. Touching the one-letter code for any amino acid displays its 3-letter code and sequence number in the slot in the middle frame. Try it.
    3. Clicking the residues in the sequence highlights their positions in the 3D structure. Try it.
    4. The show and select range option (top frame of Seq3D window) allows you to highlight a range of residues by clicking on the first and last residues of the range. Try it.
    5. In the top frame of Seq3D, scroll down. Click the checkbox to the right of the green C. Click the button [Apply Checked]. Now all cysteines in the sequence of chain A are highlighted in green.
    6. The accumulate selections checkbox allows you to select any set of residues by clicking on them. Check it, and click all 6 of the cysteines in chain A. In QuickViews, click [Center], Cancel to center the currently selected 6 residues. Zoom in.
    7. Residues highlighted with Seq3D remain selected. You can use QuickViews to change the rendering or coloring of residues selected in the Seq3D window.
       COLOR Structure. DISPLAY Dots. How many of the 6 cysteines are in alpha helices?
    8. Finally, the option Scrutinize range is provided in the top frame of the Seq3D window. This is designed to make it easy to visualize whether internal gaps in sequence numbering represent missing amino acids in the 3D model.
       1d66 has no internal gaps (only gaps at the ends). Go to the FrontDoor, and enter PDB ID Code 1fod in the slot to start a new PE session. Open the Seq3D window for 1fod. Notice the large internal gap in chain 4. Select (in the top frame of Seq3D) Scrutinize range, then click the ends of the gap (residues before and after the dots). Now you can see easily that there is a gap in the 3D structure. This is not the only kind of internal gap that you may encounter. To learn about the other kinds, click on Help in the Seq3D window.

22. Contact Surfaces are one of the most powerful features of PE. After selecting any moiety, you can see its contacts in one click. You can visualize the contacts to a single atom, one residue, a range of residues (such as one helix), a domain, a ligand, etc. The example in the QuickTour, the pairs of cadmium ions, was a very simple one to save time. Here are some richer examples.

    Contacts to an entire chain.

    1. Reset the view of a session on 1d66, or start a new 1d66 session from the first Quick-Start link on the FrontDoor.
    2. Notice that chain A makes contacts with protein and DNA, and that the latter include contacts to DNA backbone (nonspecific salt bridges) and DNA sequence-specific contacts in the major groove.
    3. In QuickViews, SELECT Chain A, DISPLAY Contacts.
    4. Center, zoom in, and examine each of the above 3 types of contacts. In the sequence-specific region, you can recognize DNA base rings. Click on the 3 that contain balls, and watch the identification reports. Gal4 recognizes CGG -- can you confirm this?
    5. Try the numerous options in the middle frame that modify the Contacts display.

       Contacts to a single residue.

    6. Restore the display of contacts to chain A (SELECT chain A, DISPLAY Contacts).
    7. In the middle frame, in the Contacts help, scroll down to the block of controls and click Backbones: Show.
    8. Open Seq3D. In the top frame, use the pull-down menu to change the display mode to "Dots". Click Cytosine 13. Notice where it sits in the overall structure.
    9. DISPLAY, Contacts. Center and zoom in.
    10. In the center frame, click these Contacts controls:
        • Surface: Dots
        • Atoms inside + outside surface: 7 Å (The point of "inside + outside" is to show covalent bonds between the atoms inside and outside the surface. In this case, note the DNA strand backbone bonds connecting to C13.
    11. You should now be able to observe:
        • Stacking of C13 with adjacent rings in the same strand.
        • Watson-Crick bonding to the opposing G26 in the opposite strand.
        • Salt bridges between two cationic amino acid sidechains and the phosphate of C13.
    12. For a nice view of the W-C hydrogen bonds, click Atoms inside + outside surface: 7 Å (to select all of the visible atoms), then DISPLAY, HBonds, Donor atoms to acceptor atoms.

23. SELECT Clicked. Sometimes you need to select something that is not on the SELECT menu. An example is just one of the two pairs of cadmium ions in 1d66 (SELECT Ligand selects both pairs). SELECT Clicked allows you to select any atom(s), residue(s), or chain(s) by clicking on them. Selected items turn orange. If you select something mistaken, just click it again to deselect it. When you are finished selecting, use the Change link in the middle frame to stop selecting by mouse clicks. Try it!

24. Distances, Angles, and Labeling. PE can report distances between atoms, angles, dihedral angles, and can attach arbitrary labels to atoms. These features all involve displaying information for atoms chosen by clicking, so they are enabled with DISPLAY Clicks. Try them!
    • Distances can be reported in the message box, or displayed on a line connecting the two atoms (a monitor line).
    • Labels remain attached to their positions when the molecule is rotated. It is often useful to put one or more spaces before the label text to space the label away from the atom to which it is attached.

25. Cation-Pi Interactions; Salt Bridges. Options on the DISPLAY menu will find and display cation-pi interactions or salt bridges. For cation-pi interactions, a link to more information in the middle frame shows an introduction, galleries of interesting examples, and tutorials for difficult cases. Try them!
    QuickViews shows cation-pi interactions and salt bridges only between amino acids. (It ignores non-amino acid components.) Advanced Explorer has cation-pi and salt bridge tools that enable you to include ligands or other non-amino acid structures in these displays.

26. COLOR Temperature highlights in "warm colors" (yellows, oranges, reds) regions of crystal structures that had the most disorder. This warns you which regions of the model have greater positional uncertainty. Try it!

27. The Molecule Information Window has links to important resources for the current molecule. Open it and get familiar with these resources.
    • In particular, the link to the Probable Quaternary Structures site of the European Institute of Bioinformatics shows you the specific oligomer for your molecule.
    • The Molecule Information Window also offers access to the header of the PDB file, which contains the primary literature citations, full names of ligands, and other crucial information.
    • The Molecule Information Window also offers methods for loading a single chain of your molecule, an introduction to crystal contacts and how to visualize them, and other resources.

28. MSA3D is one of PE's most powerful features. It colors the 3D protein model to show regions of conservation or mutation from a multiple protein sequence alignment. For an introduction, go to the FrontDoor and click on the small image at the top left, labeled Color by multiple protein sequence alignment. After reading this, if you want to try MSA3D, go to Advanced Explorer and do the MSA3D Tutorial, which includes built-in ready-made alignments.

29. PE's FrontDoor introduces many important capabilities that should not be overlooked. Go to the FrontDoor and read about structure searching, visualizing alignments between chains, making web pages with hyperlinks that prespecify molecules to be shown in PE (such as class home pages), downloading PDB files, Protein Comparator for side-by-side comparison of two molecules, etc.

30. PE has built-in capability for playing animations of protein conformational changes. These "movies" can be viewed from any rotated perspective, and in a variety of renderings and color schemes. For an introduction and several examples, go to the FrontDoor and click on the small animated image labeled EF Hand binding calcium. The same tools, the NMR Models/Animation page of Advanced Explorer, can also animate ensembles of models from NMR experiments, simulating thermal motion.

31. The Noncovalent Bond Finder provides a more detailed (bond-by-bond) visualization of noncovalent interactions, in contrast to the overview provided by QuickViews DISPLAY Contacts (covered extensively above). After you select any moiety of interest, it moves out in steps of 0.1 Angstrom and displays the shells of closest atoms. It can be restricted to show only desired categories of atoms, such as carbons in hydrophobic sidechains. It has an introductory tutorial. Access to the Noncovalent Bond Finder is at Advanced Explorer.