1-Hour Tour for Protein Explorer
(formerly called the QuickTour until it was pointed out to me that one hour may not seem "quick" to everyone)
by Eric Martz. Revised August, 2002; July 2003; June & August 2004; January 2005, June 2006.
Available on-line at proteinexplorer.org/qtour.htm. Feedback to emartz@microbio.umass.edu.

If you have not viewed the Protein Explorer Demo movies, please do so before starting the Tour below.

This 1-Hour Tour will help you learn 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.

This Tour 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 1-Hour Tour 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 Tour document.

1. If you haven't already started Protein Explorer, start a compatible web browser and go to www.proteinexplorer.org.
   (If your browser is not compatible, Protein Explorer will tell you how to make it compatible.

2. First you will see Protein Explorer's FrontDoor page. If you have not already viewed the Protein Explorer Demo movies, please do so before continuing this Tour. After you have viewed the Demo movies, proceed by clicking 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 steps E and F5 in the Troubleshooting guide. Problems common in Mac OS X/Classic can be avoided by following these suggestions.

   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, www.pdb.org). (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 in the control panel (upper left). 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. If you happen already to be familiar with RasMol commands, you can use them here, but this is not necessary at all. See also the note beneath Step 8 below.

4. Near the bottom of the FirstView control panel (scroll down) is a link to Record Your Observations. If you have two or more hours for your Tour, you may find it useful to print this and note your observations. If you want to complete your Tour in an hour or less, you can skip this form.

5. Before you leave 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.

6. When you are finished with FirstView, click the link near the bottom to "Explore More at Features". Take a few minutes to explore the Features of the Molecule control panel. At the top is important information provided by the authors of the structure. At the bottom are links that display ligands or residues designated by the authors. In the case of 1d66, only the cadmium ions are offered. Click on the link CD, accept the offer to simplify the image, and Cd ions will flash.

7. Scroll down in the Features of the Molecule control panel to find a cluster of gray buttons. Try out the Spin, Zoom+, -, Bkg, Water, and Ligand buttons (save the others for later). Click each one more than once. These convenience buttons are available on all control panels after FirstView.

8. When you are finished with Features of the Molecule, click Explore More with QuickViews. QuickViews is the heart of the user-friendly power of Protein Explorer. Feel free to try anything here that interests you. In the Tour steps below, we'll introduce you to the most important capabilities, but by no means all capabilities of QuickViews.
   Note for RasMol users only: Protein Explorer is designed to accomplish most visualization tasks quickly and efficiently from the menus, buttons, and form slots in QuickViews without learning a single RasMol command. Although you may enter RasMol commands into Protein Explorer, you will miss its major benefits unless you start out by learning to use the QuickViews menus. After you are familiar with these menus, on occasion you will be able to accomplish something more easily by entering a RasMol command -- but only occasionally!

9. Let's start with Secondary Structure. Press the gray button labeled 2^o (between the Slab and Undo buttons). You can see this image better on a black background, so press the Bkg button until the background turns black. In 1d66, notice the longest two alpha helices that are parallel.

10. Generally, use the QuickViews menus left to right (1, 2, 3; SELECT, DISPLAY, then COLOR). Notice the count of atoms selected below the molecule. Now, open the pull-down SELECT menu and click on Nucleic. Did the count of "atoms selected" change? Notice that when you select something, the molecular image does not change until you specify DISPLAY or COLOR options. These always operate on the currently selected atoms. For example, now DISPLAY Spacefill, then COLOR ACGTUbb.

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 Undo button. After each click, before clicking again (or doing anything else) wait until the green Ready indicator appears below the molecule. If you make a mistake, try the Undo button!

13. Distribution of Hydrophobic Residues. SELECT Protein, DISPLAY Spacefill, COLOR Polarity2. This is easier to see on a black background. In 1d66, notice the continuous, buried hydrophobic strip where the two longest alpha helices contact each other. This can be seen most dramatically by using the Slab button (rotate until you can see the entire strip). 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, enter the code 1bl8. Color the protein with Polarity2, and look at the protein surface for evidence of where it sits in the lipid bilayer.

14. Amino and Carboxy Termini. SELECT Chains, DISPLAY Spacefill, COLOR N->C Rainbow. Which end of the protein chain is synthesized first?
    The primary publication for 1d66 (available at Features of the Molecule, which can be accessed with the link PE Site Map near the convenience buttons) states that the intact Gal4 protein has 881 residues. As we shall see when we display the sequences (in step 17 below), only the 65 N-terminal residues were crystallized in 1d66.

15. Net Charge. COLOR +/- Charge (which is the same color scheme as Polarity5). What do you think is the net charge of the 1d66 protein? How would this support the function of Gal4? You will be able to see the protein charges better if you SELECT Nucleic, DISPLAY Backbone. Pay attention to the DNA-contacting surface of the protein.

16. At the bottom of the QuickViews control panel, click on Beyond QuickViews: PE Site Map The Site Map Window that opens shows all the control panels available in PE. You can see where you've been, and where you have yet to go. This Site Map enables you to jump anywhere at will. It is available in all control panels.

17. At the top of PE's Site Map is an important link to Help, Index & Glossary. This is the best place to go when you have questions, such as how to do something new, or where something is, or definitions of terms. This is also where you can find answers to FAQ (frequently asked questions). The Help/Index/Glossary is always available by clicking a circled green question mark () near the top of any control panel in PE. All green links in PE go to the Help, Index & Glossary.
    In the Help, Index & Glossary, click on L and read about the Limitations of PE.

18. From the PE Site Map, open Sequences. Find Chain A of 1d66, and touch residues in the one-letter code sequence. Notice that the 3-letter amino acid abbreviation and sequence number appears in the white slot in the middle of the window as you touch each one-letter code.
    There are many options in the Sequences display, and you can skip most of them for now. The most important things to look for are gaps and identical chains.

    For 1d66, notice that protein chain B is identical to chain A. The DNA chains (strands) also have identical sequences. However, different authors number DNA strands differently, and PE usually can't discern whether they are identical.

    Some X-ray crystallographic results have gaps -- typically regions where a portion of the chain could not be resolved. These are easy to spot on the Sequences display as rows of periods "....". For 1d66, the first seven residues of chain A were unresolved. (The numbering of DNA chain E starts with a row of dots, but that is an artifact of the way it is numbered, not a result of unresolved residues.) If you have time for a digression, enter PDB code 1FOD into the slot on PE's FrontDoor to start a new session. (PE allows multiple concurrent sessions, so you don't have to close your 1d66 session.) Chain 4 of 1FOD has unresolved residues both at the N terminus, and in the middle.

19. Ligand Contacts. The ability to identify noncovalent bonds easily, and see them clearly, is one of the most powerful and popular capabilities of PE. The following procedure introduces you to number of other useful techniques in PE as well.
    1. With PE displaying 1d66, open the Site Map and click Reset Session.
    2. Go to QuickViews. (From FirstView, there is a direct link below the Features of the Molecule link, or you can use the Site Map.)

       First, we'll make the ligand clearly visible.

    3. Click the Water button to hide water.
    4. Click the Bkg button to make the background black.
    5. SELECT Ligand, DISPLAY Only.

       Now, we'll center it and zoom in.

    6. SELECT Clicked, and choose "one atom per click", OK to zero. For 1d66, click on just two atoms in one of the pairs, so that 2 atoms are selected (see report in the slot below the molecule). If you are exploring something other than 1d66, other methods of selecting the moiety of interest may be easier.
    7. Click "Stop" in the top line in the middle frame.
    8. Click the Center button, then Cancel in the pop-up question.
    9. SELECT Chains, DISPLAY Backbone.
    10. Press the Zoom+ button repeatedly until you can see the ligand clearly (about 8 times for 1d66). (A shortcut for zooming faster is described in the middle help frame, after you press the Zoom+ button once.)

        Finally, let's visualize noncovalent bonding to the ligand.

    11. SELECT Clicked, and check the option to re-select the atoms previously selected by clicking (2 atoms for 1d66).
    12. DISPLAY Ball+Stick.
    13. DISPLAY Contacts. From the menu that appears in the middle frame, choose "Decorate Surface ...".
    14. Next, in the middle frame, choose step by step, then click the "Show Contacts" button.
    Rotate and 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-decorated 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. (I believe the holes in the surfaces around the cadmium ions are a bug -- not important.)

20. Using Contacts Controls. The ligand (1d66: pair of cadmium ions) is represented by its van der Waals surface. Scroll down in the middle frame to the Controls for Contact-Decorated Surfaces. Hide the surface, then click on the gray "Ligand" button (in the upper left control panel) once or twice until the cadmium ions are enlarged.
    Hint: If the Contacts help is not visible, click "Restore Contacts Help & Controls" (top of middle frame). 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? How many atoms of this element are bound to the pair of Cd ions?
    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 amino acids form a coordination cage around cadmium?

21. Adding a MolSlide. In the bottom left frame (containing the "Message Box"), scroll below the message box until you see a group of links titled "MolSlide Maker and Molecular View Recorder". Click on Save This View... In the SAVE VIEW dialog that opens, click on Add a new MolSlide .... The MolSlide Manager that opens is similar to PowerPoint, but specialized for making slides of molecular views that you can rotate. Unless you have plenty of time, we don't have time to explore its capabilities now -- it is just important that you know that Protein Explorer includes the capability of making MolSlides.
    • You can save as many views as you wish here.
    • You can add text in any language (HTML and color quick help is provided).
    • You can reorder your MolSlides, and two can be joined in to a side-by-side comparison MolSlide.
    • MolSlides can be saved (and later viewed on-line or off-line from your disk) using either Chime or Jmol. Using Jmol makes your MolSlides viewable in Safari/OSX, and on linux.
    • A MolSlide can be applied to PE. Thus, a saved MolSlide can be used to restore a previous PE session, so you can start exploring at the view you saved into the MolSlide.
    MolSlides are one of the most powerful capablities of PE. When you have time, look as some examples at http://molslides.proteinexplorer.org.
    
    To proceed with the One-Hour Tour, close the MolSlide Manager.

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

23. Placing Contacts in Context. If the Contacts help is not visible, click "Restore Contacts Help & Controls". Scroll in the middle frame until you find the Controls for Contact-Decorated Surfaces. 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.

24. History. In the bottom left frame (containing the "Message Box"), scroll below the message box until you see a group of links titled "MolSlide Maker and Molecular View Recorder". Click on History... The RECORDED HISTORY dialog that opens enables you to view any earlier image in your session. If you wish, you can apply an earlier view to PE -- but notice that reverting to an earlier view cannot be undone.

25. DNA vs. RNA. Open the PE Site Map, and select Reset Session. When the FirstView is restored, 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.

• Browse the Atlas of Macromolecules (linked in the left gray box on the FrontDoor of Protein Explorer).
• To find and display a molecule of particular interest to you, go to 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 Tour, and are ready to try out more capabilities of Protein Explorer (www.proteinexplorer.org).

In order to restrict it to 1-2 hours, the above Tour 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.

The 1-Hour Tour is designed to be printed on paper, so hyperlinks were minimized. However, the section below has many useful hyperlinks so if this copy is on paper, you may prefer to use it on-line (click on 1-Hour Tour at the FrontDoor of PE, and scroll down to this section).

26. MolSlides are one of the most powerful capabilities of PE. Visit MolSlides.ProteinExplorer.Org for an introduction, and examples.

27. 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 PE Site Map, and in it, 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. Click the large gray button at the top of the Seq3D window Show all as thin backbones, then try clicking some arbitrary residues.
    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. How many of the 6 cysteines are in alpha helices?
    8. It took some effort to select the 6 cysteines. Use SELECT Saved to save the current selection. Now you can re-select it later with much less effort!
    9. 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.

28. Contact-Decorated 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 1-Hour Tour, 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. From the menu that appears in the middle frame, choose "Decorate Surface ...".
    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, Decorate surface ...).
    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.

29. SELECT Clicked. Sometimes you need to select something that is not on the SELECT menu. An example used above in the 1-Hour Tour was to select only 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 Stop link in the middle frame to stop selecting by mouse clicks. Try it!

30. 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.

31. 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.

32. 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!

33. From the PE Site Map, the External Resources Window has links to important information about the current molecule. Open it and get familiar with these resources.
    • In particular, the link to the Probable Quaternary Structures (PQS) site of the European Institute of Bioinformatics shows you the specific oligomer for your molecule. For example, 1K4C contains an antibody fragment (Fab) bound to the potassium channel. But what you get in file 1k4c.pdb (the asymmetric unit) is only one quarter of the channel. PQS offers you the complete tetramer.
    • External Resources also offers methods for loading a single chain of your molecule, an introduction to crystal contacts and how to visualize them, and other resources.

34. ConSurf (consurf.tau.ac.il) is a very powerful tool. It colors a protein to identify the most conserved and most rapidly evolving residues according to a multiple protein sequence alignment (which ConSurf will construct entirely automatically). The result is displayed in Protein Explorer. Take a look at ConSurf's Gallery for some examples, and then try a molecule of interest to you. ConSurf usually returns results within a few minutes.
    • From within PE's QuickViews, you can get to ConSurf with DISPLAY Evolution. This produces a link to ConSurf in the middle help frame. ConSurf is also linked to PE's FrontDoor -- click on the small image labeled Color by multiple protein sequence alignment. Advanced Explorer (available in the PE Site Map) includes an earlier method to make a similar color scheme, called MSA3D. For most purposes, ConSurf is not only much easier to use than MSA3D, but its results are more meaningful and robust.

    • You can download a single PDB file containing the results of any ConSurf job. Look for the Download link near the bottom of PE's ConSurf control panel. After saving the file to disk, use the Empty Explorer link on the FrontDoor, then Browse to display the saved file. PE will automatically recognize this PDB file as a ConSurf result, and display it with ConSurf controls. Bundled with PE are some Examples of downloaded ConSurf runs.

35. PE's FrontDoor introduces many important capabilities that should not be overlooked. Go to the FrontDoor and read about finding PDB files, making web pages with hyperlinks that show molecules you specify in PE (such as class home pages), downloading PDB files, displaying saved PDB files with Empty Explorer, etc.

36. 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.

37. Power users will appreciate the Preferences options, especially turning on expert mode. Preferences remain in effect between sessions. The command language can be learned by observing the commands generated by operations in QuickViews, displayed in the message box. Those who like using commands will enjoy the abbreviated command aliases, and the shortcuts called Commands to Protein Explorer (as distinct from "to Chime").