Tutorial for Protein Explorer version 1.72,
with Tutorial Study Questions.

by Eric Martz, copyright © 2000
See conditions for use.
If you are reading a paper version of this document, it is also available at http://www.umass.edu/microbio/chime/explorer/pe_tut.htm
Thanks to Frieda Reichsman for numerous insightful suggestions.
Please Note: You do not need to do this tutorial in order to use Protein Explorer, which is designed to be self-explanatory.
This tutorial is for people who have tried Protein Explorer (PE), completed the 1-Hour Tour, consulted the Help, Index & Glossary, yet want a more comprehensive tour of PE's capabilities. Many hours are needed to do all of this Tutorial. It is the basis for a Molecular Visualization Lab Course.

You can also use the Tutorial as a reference manual. To find out more about just one topic, use the Contents below.

This tutorial was last revised in fall, 2000, for PE version 1.72. Therefore, it does not yet incorporate new features of PE such as the 1-Hour Tour, the new Animation capabilities, several enhancements in Contact Surface displays, the new integration with EBI's Probable Quaternary Structures site, new information on crystal contacts, the pI server now linked under the Polarity5 color scheme, SELECT Clicked (in QuickViews), or the existence of the Help/Index/Glossary or FAQ.

New advanced features not incorporated below include the introduction to homology modeling and methods for mutating residues with DeepView,

The "Features of the molecule" control panel (introduced in PE 2.1, July 2003) is not incorporated in this tutorial.

The tutorial does not know about using the "PE Site Map" (introduced in PE 2.1, July 2003) to navigate within PE.

 

Contents

 

Background.

    HIV protease with Q8261
    inhibitor bound (1HVH).
     
  1. What is the Protein Explorer good for? The Protein Explorer (PE) enables you to view and explore the three-dimensional (3D) structure of any macromolecule. You can explore proteins, DNA, RNA, carbohydrates, and complexes, such as between transcriptional regulatory proteins and DNA, or enzymes and drugs. There are many excellent websites which teach 3D structure of particular molecules (see the list at the World Index of Chime Resources). The difference is that the Protein Explorer allows you to choose which molecules you wish to view and explore from among the thousands for which 3D structural data are available.  

  2. How easy is PE to learn to use? Beginners can use PE immediately, without reading this tutorial, because PE has extensive built-in context-triggered help, and a logical yet powerful QuickViews menu system. PE is much easier to use than RasMol, and much more powerful. Advanced users may wish to learn some of the command language common to RasMol and PE (see How is PE related to RasMol? below).  

  3. Can I really look at any macromolecule? PE cannot show you a protein if all you have is the amino acid sequence. In order to show you a macromolecule, PE needs structural data for that molecule. Specifically it needs an atomic coordinate (or "PDB") file (see www.umass.edu/microbio/rasmol/pdb.htm for details) and such files are available for several thousand different molecules (more about how to get them in Chapter IV below). Since there are about 80,000 different proteins in humans, and since many of the structures available represent fragments, rather than entire protein molecules, there are many proteins for which no accurate atomic coordinate data are available. Most macromolecular structures are determined experimentally by X-ray crystallography. See Origins and Limitations of 3D Structural Data. In particular, transmembrane domains of proteins cannot be crystallized, so few examples are available. Despite this, structures are available for a large number of important proteins. 3D structure cannot be accurately predicted ab initio from amino acid sequence. However, when an experimentally determined structure is available for a sequence-similar protein, a reasonably reliable structure can be predicted by homology modeling.  

  4. What do I need to know before using PE? This tutorial assumes that you have some knowledge of general and organic chemistry, amino acids and proteins. If you are unfamiliar with some of the concepts and terms used below, consult any textbook of biochemistry.  

  5. What is a good book on 3D protein structure? Any recent biochemistry textbook will include an introduction to 3D protein structure. For a broader overview, I recommend the short, excellent, readable and lavishly illustrated Introduction to Protein Structure, second edition, by Carl Branden and John Tooze, Garland Publishing, 1999 ( www.garlandpub.com/SCIENCE/302703.html).  

  6. How is PE related to RasMol?. PE is an enhanced derivative of RasMol. Persons familiar with RasMol will find PE's menus similar, and all RasMol commands are understood by PE. More importantly, persons who have never used RasMol will have no trouble using PE. PE version 2.0 is much easier to use and much more powerful than RasMol. RasMol is the cross-platform freeware by Roger A. Sayle which brought high-quality image rendering and fast rotation of macromolecular structure to the personal computers of the masses in 1993. PE depends upon MDL's Chime, a Netscape plugin which incorporates source code from RasMol for image rendering and command processing. Chime (CHemical mIME), by Tim Maffett, Bryan van Vliet, Franklin Adler, Jean Holt and others at MDL Information Systems, Inc., retains all of RasMol's menu functions and command language (with the exception of writing GIF image and vector postscript files, and direct writing of scripts to files), and includes many powerful enhancements (such as surfaces). PE is a "wrapper" for Chime that makes the power of Chime more accessible. See the Purpose of the Protein Explorer for more of the history and rationale. For more on the history of molecular visualization, see the RasMol Home Page www.umass.edu/microbio/rasmol), especially www.umass.edu/microbio/rasmol/history.htm, and www.umass.edu/microbio/rasmol/rasintro.htm.  

  7. Can I use RasMol tutorials with PE?. Yes you can, since PE understands the complete RasMol command language. However, because PE is so much easier to use, you should complete Chapter I of this tutorial first, so you know how to use PE. Then, advanced users may wish to try some RasMol tutorials in order to have a guided tour of specific molecules, while learning more about the RasMol/Chime command language. See Chime & RasMol: Software Training for Faculty and Students at www.umass.edu/microbio/rasmol/softhelp.htm.
 

Start Protein Explorer

     
  1. How is this tutorial organized? The first three chapters of this tutorial introduce the three levels of Protein Explorer: FirstView, QuickViews, and Advanced Explorer. You will explore specific example molecules, especially 1d66.pdb, a protein:DNA complex. Chapter IV discusses how to find macromolecules at the Protein Data Bank, and general instructions are given for exploring any molecule of your choice.  

  2. Learn by doing! You will learn best if you do the steps below in the Protein Explorer (PE) while you are reading this tutorial. This can be done either (i) by printing this document, or (ii) by switching back and forth between the tutorial window and the PE window in your browser. (When you click on Tutorial in PE, it opens a second window.) You may wish to use both methods at once, so you can have the advantages of working from paper, yet also click on the hyperlinks in the tutorial whenever you wish.  
  3. Tutorial Study Questions. A list of study questions is available to accompany this tutorial. Answer the questions as you work through the tutorial. You can print the questions on paper and write in your answers. Or you can use Netscape's File, Edit Page menu option to type your answers in Netscape Composer. Then be sure to use Netscape Composer's File, Save As menu option to save a copy of your answers to disk!  

  4. Start PE. From now on, this document will refer to what you are seeing and doing in the PE window. Start PE by typing this URL into the "Location" slot of your browser: proteinexplorer.org. The first screen has lots of information that we can skip for now -- but bookmark it!. Just click on Quick-Start Protein Explorer. (If you have not installed Chime, or if you are not using a compatible browser, you will be given installation instructions at this point.)  

  5. Maximize your window size. Carefully read the screen which begins "If this window is not the size you want ...", and resize the window appropriately. Notice the default Session Name. This will appear at the top of your PE window, and on the window's button on the task bar (Windows) or on the Communicator menu (Macs). You can change it if you want. It is possible to run multiple PE sessions concurrently. The session number helps to identify each session. Click the big link Start Explorer Session and be patient (your browser must digest thousands of lines of javascript at this point!). After PE loads, your browser window will be divided into 3 frames. A molecule for use as an example in the tutorial will load automatically. At the top left it should say FirstView: 1d66.pdb.  
  6. Proceed only when PE is ready. Notice the Busy/Ready indicator below the molecule. Whenever you click on a button or link, or issue a command, the red busy warning will come on. After a few moments, it will change to the green ready signal, . As you proceed below, keep an eye on this indicator; don't push buttons until PE is ready. Clicking buttons before PE has caught up has been known to confuse PE (making it permanently busy). If this happens, just restart it -- no damage is done other than losing your session.
 

Chapter I: FirstView

     

  1. FirstView reveals the answers to a lot of fundamental questions about your molecule -- if you know what to look for and how to interpret it. Whenever you look at a new molecule for the first time, spend enough time here to get the most out of FirstView. This Chapter explains how to do that.  

  2. How can I find help quickly? The link Protein Explorer © 2000 by Eric Martz: is present at the tops of all pages in PE and provides convenient access to the main help system, including this tutorial.  

  3. Why is the molecule spinning? By default, PE shows molecules spinning when first loaded. If the molecule is not spinning, click [Toggle Spinning]. Spinning helps greatly to appreciate the 3D structure. However, automatic spinning also keeps your computer fairly busy and slows down PE's responsiveness to buttons or menu selections.

    • Throughout this tutorial, we'll indicate buttons by enclosing their names in [ ], for example will be indicated as [Toggle Spinning].
    • Turn off automatic spinning when not gazing transfixed at the beauty of the rotating molecule. PE will respond more quickly when spinning is off.

     

  4. How can I see the molecule from different perspectives? With spinning off, drag on the molecule with your mouse. Dragging rotates the molecule about the X and Y axes. You should rotate the molecule with the mouse early and often to help explore the molecule. In most situations, this makes stereoscopic viewing unnecessary.

    Rotate the molecule with your mouse early and often!

     

  5. What does 1d66.pdb mean? The title at the upper left of the window is FirstView: 1d66.pdb. Each published 3D macromolecular structure is assigned a 4-character identification code by the Protein Data Bank (PDB), where all published structures can be obtained (more about this in Chapter IV). The PDB identification code for the first Quick-View molecule is 1d66. The file 1d66.pdb contains the experimentally-determined atomic coordinates for a complex between the DNA-binding domain of the yeast transcriptional regulator protein Gal4, and a short palindromic segment of DNA. Its structure was determined by X-ray crystallography.

    "PDB" refers not only to the Protein Data Bank, but also to a data file format in which the atomic coordinates for a 3D macromolecular structure can be stored. The PDB format (see www.umass.edu/microbio/rasmol/pdb.htm for details) is one of the most commonly used formats for atomic coordinate files. In order for PE to display a molecule, it must be given an atomic coordinate file. Such a file in the PDB format for 1d66 has the standard filename "1d66.pdb".

     

  6. What are all those red spheres? The initial image shows a large number of red spheres. Click on one of them and watch the identification report which appears in the message box (in the lower left frame of the screen). Click the button [Hide/Show Water] several times. Click the water link in the FirstView panel, or the framed picture of a water oxygen atom, to find out more about water in PDB files.

    Click on it!
    Whenever you're not sure what an atom is, click on it to get an identification report.

     

  7. How many chains are there? Click the link backbone trace on the FirstView page to review this topic. At the bottom of the FirstView page is a link Form for Recording Observations. Obtain a copy of this form on paper, or print one now. Fill in the first column with the names of the chains in 1d66.  

  8. Is there anything else in this PDB file besides the protein/DNA chains? With water hidden, four magenta spheres remain visible in addition to the backbone chains. Click on one and the report will tell you that it is a cadmium atom. The physiologic metal for the Gal4 DNA-binding domain is zinc; Cd was used however in the preparation of the crystal resolved here. Click on the link hetero atoms on the FirstView page and read this document.

    FirstView
     PE's FirstView shows you crucial information:
    • Protein, DNA or RNA chains as backbone traces, each chain a different color,
      hence, the number of chains.
    • All other moieties ("hetero" atoms) spacefilled, such as
      carbohydrates, ligands, metals.
    • Water if present.
    • Disulfide bonds if present.
    • Anything else in ball and stick.

     

    Chapter II: Molecule Information Window, PDB Header, & Sequences

     

  9. Molecule Description and PDB Header. Click on the red and yellow molecule information icon , which is visible on any control panel in PE. The Molecule Information window should appear.

    At the top is a brief description of the molecule. This is obtained by Protein Explorer from information in the PDB file header (HEADER and COMPND records). (PDB record names are limited to 6 characters.) The PDB header is the portion of the PDB file prior to the beginning of the atomic coordinates (ATOM or HETATM records). In some PDB files, there are multiple COMPND records (lines), in which case the Molecule Information window shows only the first line (due to a limitation in Chime).

    Clicking the PDB File header link will display the entire header, obtained from the PDB website. Note whether there is more than one COMPND record. Other important information is the deposition date (top line), the organism (SOURCE records), the authors and literature citations, and the resolution in REMARK 2. The uncertainty of the position of an atom is roughly one fifth to one tenth of the resolution for high-quality data (R-factor 0.20 or less, succinctly explained on p 160 by Rhodes). For NMR results or theoretical models, resolution values are not applicable, and so not given. The HET, HETNAM, and FORMUL records are very useful to figure out what the cryptic 3-letter residue codes mean for hetero residues (see next section below). The HELIX and SHEET records are used by Chime to display secondary structure cartoons and colors.

    An important record is EXPDTA, which tells you the method for determining the coordinates. 1d66 lacks an EXPDTA record. There is a large effort underway at the PDB to clean up the "legacy" PDB data to make the files uniform and their contents more machine readable, but many files have not yet been processed.

    You can enter any PDB ID code into the slot in the Molecule Information Window, and these links will fetch the corresponding header. Enter 1BL8 and view its header -- it has the EXPDTA record. Notice that it has multiple COMPND records.

    Use the Molecule Information Window!
    You are expected to use the Molecule Information Window whenever you load a new molecule and need to know more about it. From now on in this tutorial, we won't remind you.

     

  10. Identifying HETERO compounds.

    When you click on a hetero group, its name is messaged. (We'll use "messaged" to mean "displayed in the message box" at the lower left.) However, these names are limited to 3 characters and are usually cryptic. The first place to check is the PDB file header, namely the records HET, HETNAM, and FORMUL.

    A good place to find out more is the Hetero-compound Information Centre - Uppsala of Gerard Kleywegt (HIC-Up). Click on the tiny link Search this site at the left, then the link (under item #2, Search Tips) QuickXS pop-up menu. Enter the 3-letter code in the slot under QuickXS II. In the result, scroll to the bottom to see a 2D structure as a GIF. Other useful links are the MDL Chime page (scroll to the bottom to see the hetero compound in Chime), and list of PDB files containing this compound.

    If you should be so unlucky to encounter a hetero compound with a one-letter or two-letter name, HIC-Up won't work. Go to PDBSum (University College London), enter the PDB ID code, then at the very bottom of the result page, see the links for the hetero compounds.  

  11. RCSB's Structure Explorer.

    Re-enter 1d66 in the slot at the Molecule Information Window, and look at RCSB's Structure Explorer page.

    An important link is the Medline link which allows you to read the abstract (and in some cases, full text) of the original article.

    Although they are hard to see on the blue background, the links down the left side are very powerful.

     

  12. Sequences. Press the Sequences link (not Seq3D). Take the time to become thoroughly familiar with the information on this page, try out the coloring tools, and take a look at the help linked to it. (We'll postpone using Seq3D until later.)  

  13. Show Counts. Finally, inspect the block of counts messaged by the link Show counts (in the Molecule Information Window). This is the same information messaged when the molecule first loads, but the Show counts link brings it back when needed. Beware that the number of chains reported is usually incorrect (revisit the backbone trace help at FirstViews). The number of "chain" atoms is followed by the number of "hetero" atoms in parentheses. Ditto for groups. Note that the count of atoms for 1d66 below the molecule ("All 1,762") is the sum of the two messaged atom counts. "Group" is used in Chime as a more general term for "residue". Water oxygens and metal ions count as hetero "groups" and each atom is given a group (residue) number, just as for amino acids.  

  14. PE's Lack of Access to Header Information. Unfortunately, although the PDB file header is in Chime's memory after the file is loaded, no mechanism was provided that gives access to this information by e.g. PE. Particularly important information such as SITE records is presently inaccessible (see 4csm for a good example with regulatory and active sites described and identified). A plan is underway to remove this limitation. (For information on how to find PDB files with SITE_DESCRIPTION records, see the comparison of OCA with SearchFields at the PDB Lite site.)
     

    Chapter III: QuickViews

    From the FirstView page, click on Explore More, which takes you to QuickViews. QuickViews is a menu system with extensive context-triggered help. It enables powerful visual exploration entirely from menus. Before QuickViews became available in summer, 2000, this kind of exploration required learning a large number of teletype-style commands ("RasMol command language").  

  15. Buttons. The first thing to get familiar with on the QuickViews page is the block of buttons (below the SELECT, DISPLAY, COLOR menus). This block is available on all control panels in Protein Explorer to provide convenient access to these frequently needed functions. Try all the gray buttons and get clear what they do, except the [Stereo] and [Slab] buttons.
    1. Try [Center], OK, and click on an atom near one end of the molecule. Now rotate and zoom, noticing how the chosen atom remains centered. Now click [Center] again, and press Cancel, noticing that the entire molecule is re-centered.
    2. Don't spend time on Stereo because it is not important. Stereoscopic viewing of a split-image is much more difficult for some people than others. If you can do it easily, go ahead and enjoy it, but it isn't worth spending a lot of time struggling with stereo.
    3. Slab will be introduced later.
     

  16. Access to FirstView. Notice the FirstView links. Info Only is useful when you want to review information on the FirstView description, or access any of the many help pages linked there. Reset View reloads the PDB file and restores the FirstView image. QuickViews is the only control panel in Protein Explorer with access to FirstView.  

  17. Order of Menu Operations. Using the QuickViews menus generally requires that you first select a subset of the atoms, then display them in the desired manner (or hide them), and finally color them. It is important to realize that selecting atoms has no effect on the image until a display or color scheme is applied. After a select operation, always check the number of atoms selected, displayed in the slot below the molecule. If zero atoms were selected, display and color operations can't change the image!  

  18. Messages vs. Commands. The message box is the box below the [Clear] button, in the lower frame. With the exception of the show counts message, you do not need to watch the messages in the message box, which is provided primarily for advanced users. In particular, don't misinterpret commands shown in the message box as counts. For example, 1d66 has no disulfide bonds, which you can verify with show counts. In contrast, when you DISPLAY SSBonds (try it), the message "ssbonds 0.5" is a command that specifies that any disulfide bonds present shall be shown as bonds with a radius of 0.5 Angstroms -- not a count of disulfide bonds.

    Windows only: it is OK to drag the top edge of the frame containing the message box down, to make more room for the middle help frame of QuickViews.
    Macintosh: dragging frame boundaries in PE usually causes Netscape to freeze or crash.

     

  19. Secondary Structure: Where are the alpha helices and beta strands?

    1. Notice the number of "atoms selected" below the molecule. Initially, all atoms are selected. (You can always verify this by clicking All on the SELECT menu.)
    2. Open the SELECT menu and click on Protein. Notice that fewer atoms are now selected (not water, not DNA, not ligand).

        Notice that nothing changed in the image.
        This is an important principle.
        After selecting, you must specify how to render and/or color the selected atoms.

    3. Open the DISPLAY menu and click on Cartoon.
    4. Open the COLOR menu and click on Structure (this refers to Secondary structure).
      Now the protein is colored to indicate secondary structure. (There are no beta strands/sheets in 1d66. It is coincidence that the cadmium ions are the same color as alpha helix.) Often this color scheme is best viewed on a black background (click on the [Bkg] button until you get a black background).

        (Chime has no built-in secondary structure assignments or color schemes for nucleic acids.)

      Read the QuickViews Help!
      By now you have noticed that every time you use one of the QuickViews menus, information about your choice appears in the middle frame. This information is often quite important, but there is no reason to repeat it here in the tutorial, so be sure to read it as you try each new menu option!
      Below, you will not be reminded to read this help, and it will be assumed that you have read it.

    5. Follow the COLOR Structure instructions in the middle frame to force Chime to make its own assignment of secondary structure. Notice the appearance of light blue "turns" not present before. Also you'll notice that the ends of the helical cartoon ribbons are now white, meaning that Chime made a more conservative assignment of helical regions than did the authors of the PDB file 1d66. Chime's assignment is objective, while the assignments of authors may be partially subjective. Notice that the two protein chains are no longer showing identical secondary structures.

      Bear in mind that a real protein in aqueous medium at body temperature is vibrating a great deal from thermal motion. This means that some portions of alpha helices may fit the criteria for "alpha helix" at one instant, but not at another.

      Also bear in mind that the DNA-binding domain crystallized for 1d66 is not the entire protein. The abstract of the paper (obtained by raising the Molecule Information Window, clicking on Structure Explorer, then the Medline link) describes the protein in 1d66 as a "65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4". In fact, the complete protein is 881 residues long! The presence of the missing C-terminal 816 amino acids may influence the secondary structure in some regions of the fragment in 1d66.

     
  20. Where are the N and C termini?
    SELECT All
    DISPLAY Cartoon,
    COLOR N->C Rainbow.

    Read the help in the middle frame. Now, you can tell which end is which, and it is easier easier to trace visually the chain sequence thorough folded domains. Colors are assigned globally by residue number so that only the longest chain has red and blue ends. Shorter chains will begin and end with colors assigned to the same residue numbers in the longest chain. In this case, the DNA chains are shorter than the protein chains. Notice that the 2 DNA chains are numbered consecutively, not independently (but there are 3 different ways DNA double helices may be numbered in PDB files).  

  21. Where are the hydrophobic amino acids?
    SELECT All
    DISPLAY Spacefill
    COLOR Polarity2
    Use the [Water] button to hide water.
    Optional:
    Now you can distinguish the hydrophobic sidechains (gray). In soluble proteins these tend to be buried (away from the surface and the water contacting the surface).

     

  22. How can we see inside the protein?
    Press the [Slab] button.
    See the figure below for a brief explanation of the result.
    Rotate the molecule, carefully inspecting the two long alpha helices that don't touch the DNA.

    Also try COLOR Polarity3 and Polarity5.

    Slabbing in Chime.

    1. The molecule is cut through the center.
    2. Half of the molecule is "thrown away" (hidden).
    3. We look at the cut face, called the "slab plane". (In this illustration, we would have to rotate the cut molecule 90 degrees around the Y axis to get view 3.)

    In Chime, unlike in the figure to right, the slab plane is always parallel to the screen, and it is the portion in front of the slab plane which is hidden. In Chime, one sees the cut face and all atoms behind it (view 3 at right).


    The term "slab" as used in RasMol/Chime is somewhat of a misnomer.  

  23. How can I distinguish DNA from RNA?
    1. SELECT Nucleic.
    2. Click the link to distinguish DNA from RNA and read the help.
    3. Answer the question on DNA vs. RNA.
     

  24. Solvent Accessible Surfaces. The next few items employ solvent-accessible surfaces, so we'll start with an introduction to such surfaces. Chime (but not RasMol) has a built-in mechanism for calculating and displaying these surfaces. To understand better what these surfaces represent, we'll start with something simple: the surface of a dipeptide.
    1. Dipeptide movie. Click this link to start a new PE session that runs movie #1. (The movie is finished when returns). The movie highlights two amino acids from one chain in 1d66. Alpha carbons are colored green. Answer the question on the dipeptide movie.
    2. When the movie finishes, 17 atoms are selected (the dipeptide). If you have selected something else, close the movie window and rerun the movie. The steps below assume you have 17 atoms selected.
    3. DISPLAY Surface.
        This is the solvent-accessible surface of the dipeptide.
    4. DISPLAY ", Transp. (item below Surface; the " means "ditto", so this means "Surface, Transparent")
        Now you can see how the solvent-accessible surface relates to a ball-and-stick model.
    5. DISPLAY Spacefill
        Notice how closely the surface fits the van der Waals radii used in a spacefilling model. The depressions in the dipeptide which are hidden by the surface are too small for water to penetrate and contact them. Hence, this surface is called a water (or solvent) accessible surface. It is defined by rolling a spherical probe over the spacefilled surface. The probe has a radius of 1.4 Angstroms, which is the average radius of a water molecule.
    6. SELECT All
    7. DISPLAY Backbone
    8. COLOR Chain
    9. Use the zoom [-] button many times until you can see how the dipeptide relates to the entire 1d66 complex.

    10. Close the dipeptide session, and return to, or start a new session for 1d66. If not a new session, click on FirstView: Reset View, and hide water. (This avoids a bug I haven't had time to fix.)
    11. SELECT Chain A
    12. DISPLAY Surface showing the solvent accessible surface of chain A, if it were in this conformation in the absence of the other chains. Bear in mind that not all of the surface portrayed is in fact solvent accessible -- portions are covered by Chain B or DNA, and hence not solvent accessible. The reduction in solvent-accessible surface is an important quantitative characteristic of quaternary interactions. We'll have a better visualization in the next step below.
     

  25. Contact Surfaces: Does the Gal4 DNA-binding domain recognize a DNA sequence?
    1. SELECT Chain A
    2. DISPLAY Contacts
      Now Chain A is shown as a solvent-accessible surface, colored by distance from everthing else. You can spot a few waters hydrogen-bonded to Chain A (red atoms not connected by covalent bonds to any other atoms).

      The balls and sticks are everything not chain A, noncovalently bound to it, including DNA and chain B. Locate three regions of contacts: chain B, DNA backbone (phosphates), and DNA bases.

    3. Scroll down in the top QuickViews frame until you see the QuickViews Plus Options. Select "New display is added to previous display".
    4. Read the middle help frame carefully. Among other things, it describes a shortcut for using these Plus Options.
    5. Set Thickness of backbones and traces to 0.01.
    6. Scroll back up and SELECT All.
    7. DISPLAY Backbone
    8. Enter the command "co bb green". The first two words are aliases. Notice that the message window shows that the aliases were expanded to give "color backbone green". (Click on the Aliases link below the message window for more information.)

    9. Answer the study question "Regarding the contact surface for chain A of 1d66".
     

  26. Contact Surfaces: What holds the Cd ions in place?
    SELECT Ligand DISPLAY Contacts DISPLAY Center Optional:  

  27. Seq3D: Locating residues from sequence positions. Press the molecule information icon, and then click the link to Seq3D. The display here is similar to the Sequences display, but more compact (so you can also see the molecule). Seq3D has three primary uses, demonstrated in this and following two numbered items.

    Locating residues in 3D from their sequence positions is the default function of Seq3D, "Show clicked" (see radio buttons at top of Seq3D). For this purpose, it helps to press the button [Show All as Backbones] first, to simplify the view. Now, with 1d66, try clicking a few residues in the sequence in the lower panel. Try changing the display menu ("Show clicked residues in") to Spacefill, and then clicking additional residues.

    Whenever you rotate the molecule with the mouse, the Seq3D window disappears behind the main Protein Explorer window. To bring it back into view: On Windows, click the Seq3D button on the taskbar; on Macintosh, use the Communicator menu.

      Where are the 4 catalytic site residues in 1AI4?
    1. Start a new PE session for 1AI4.
    2. Read the abstract of the primary article on 1AI4 (Medline link on the RCSB Structure Explorer page).
    3. Examine the PDB file header, and note down the 4 residues it indicates to be in the catalytic site. (SITE record for site CAT).
    4. Bring up the Seq3D panel.
    5. Press [Show All as Backbones]
    6. Press [Ligand] to show the ligands.
    7. Click on the 4 catalytic site residues. Notice where they are positioned relative to the substrate analog.

      How are the catalytic site residues are bound to the HAA ligand?

    8. SELECT Residue, follow instructions to select HAA.
    9. Center HAA.
    10. DISPLAY Contacts
    11. On Seq3D, change the display to Dots, and check Accumulate selections.
    12. Again click the 4 catalytic site residues.
    13. Answer the question on 1AI4 in the Tutorial Questions.

      Optional to help you see noncovalent bonding relations:

    14. DISPLAY *Hide*
    15. Again select HAA.
    16. DISPLAY Ball+Stick
     
  28. Seq3D: Selecting arbitrary residues or residue-ranges. We're going to use Seq3D to select a sequence motif of functional importance, and highlight it in red.
    1. Start a new PE session for 1OSA.
    2. In the PDB header, notice REMARK 4 and the SITE records.
    3. Open Seq3D.
    4. Select "Show range".
    5. Click the first and last residues in the first EF hand "EF1".
    6. Close Seq3D.
    7. COLOR Red
    8. SELECT All
    9. DISPLAY Backbone
    10. Click [Ligands] to redisplay the metal ions.
    11. Answer the questions on 1OSA.
     
  29. Seq3D: Scrutinizing sequence gaps. Answer the study question "Are these gaps physical or virtual?".  

  30. Where are the hydrogen bonds? There are four ways to visualize hydrogen bonds.
    1. Chime's built-in "hbonds" display (protein backbone/nucleotide Watson-Crick only)
      How long is a hydrogen bond?
    2. Contact Surfaces
    3. The Noncovalent Bond Finder
    4. External assignment of hbonds
     

    (I) Chime's built-in "hbonds" display shows only the protein backbone-to-backbone hydrogen bonds in regions of recognizable secondary structure, plus Watson-Crick nucleotide base-pair hbonds. It shows none of the hbonds involving sidechains, no hbonds between chains, and no hbonds involving water. To see the bonds it does display:

      Protein hydrogen bonds. With a 1d66 session, in QuickViews:
    1. FirstView: Reset View and hide water.
    2. SELECT Protein
    3. DISPLAY HBonds, checking the second option (backbone to backbone). Make sure you read the help and understand the limitations of this display!

    4. DISPLAY *Hide* (to hide the hbonds).
    5. SELECT Chain B
    6. DISPLAY Only
    7. DISPLAY Ball+Stick
    8. COLOR Element (CPK)
    9. SELECT Helices
    10. DISPLAY Only
    11. Center the C-terminal helix (the one with residues 50 and higher) and zoom in.
    12. SELECT Chain B
    13. DISPLAY Hbonds, check the second option (backbone to backbone). Observe.

    14. SELECT Backbone
    15. DISPLAY Only (hiding the sidechains).
    16. SELECT Alpha C
    17. COLOR Green. It is very clear now that the hbonds are portrayed as white bonds connecting alpha carbons.
    18. SELECT Chain B
    19. DISPLAY Hbonds, check the first option (donor to acceptor).
    20. Answer the question about Chime's built-in hbonds display.

    21. Start a new PE session that runs movie #2. (The movie is finished when returns). Answer the question "Real bonds vs. backbones and backbone-to-backbone hydrogen bonds".
    22. Close the movie session.

      Turning to DNA:

    23. FirstView: Reset View and hide water.
    24. SELECT Nucleic
    25. DISPLAY Only
    26. Center all selected atoms.
    27. DISPLAY Hbonds, check the second option (backbone to backbone). Observe.

    28. DISPLAY Ball+Stick
    29. COLOR Element (CPK). Observe.

    30. DISPLAY Hbonds, check the first option (donor to acceptor). Observe.

    31. SELECT All
    32. DISPLAY *Hide*
    33. DISPLAY Hide Sel.
    34. Open Seq3D, check "accumulate selections", and click on DNA residues 13 and 26.
    35. Close Seq3D.
    36. Center the selected atoms, zoom in.
    37. DISPLAY Hbonds, backbone to backbone.
    38. Answer the question about Three hbonds.

    39. Repeat the above block of operations, selecting instead residues 12 and 27.
    40. Answer the question about Two hbonds.

    41. Repeat the above block of operations, selecting instead residues 11 and 28.
    42. Answer the question about the longest hbond.
     
    How long is a hydrogen bond?
    Average length of a hydrogen bond. Actual hbonds vary between 2.5 and 3.5 Angstroms.
    1. Scrutinize the base pair resulting from the last of the previous steps. (Or regenerate that image by opening a new session to play movie #3.) Remember that in a crystallographic result with the resolution of 1d66 (2.7 Angstroms), hydrogens cannot be resolved. In 1d66, none were modeled in. (Optional: See the for base pairs which include hydrogens.) Average distances are shown at right; donors are usually 2.5 to 3.5 Angstroms from acceptors.

    2. DISPLAY Clicks
    3. Select "Report distances".
    4. Answer the question "Regarding the 3 hbonds between G11 and C28 in 1d66, as Chime depicts them (movie #3)".
    5. Click the link Change in the middle frame, and restore mouse clicks to identifying atoms.

      (B) Contact surfaces. A contact surface, colored by distance, gives a useful overview of all polar and hydrophobic interactions between any two arbitrary groups of atoms. While hydrogen bonds are not shown as bonds, their positions can be deduced from the proximities of donors and acceptors near the contact surface. Previously in this tutorial we used contact surfaces to answer Does the Gal4 DNA-binding domain recognize a DNA sequence? and to see What holds the Cd ions in place? Later in this tutorial, under Advanced Explorer, we will see how to display transparent contact surfaces showing the proximal atoms on both sides.

      (C) The Noncovalent Bond Finder (NCBF). Also accessed from Advanced Explorer, the NCBF allows a detailed, distance-based exploration of hydrogen bonds. Again, NCBF does not display hbonds as bonds, but leaves their assignment to your judgement based on donor-acceptor distances.

      (D) Assignment of hbonds by external programs. A number of programs are available on the web that can calculate the positions of hydrogen bonds. A planned enhancement is to enable one or more of these programs to display hydrogen bonds in PE. One such program is the HBPLUS routine of Thornton and McDonald.  

  31. How can I see salt bridges and cation-pi interactions?
    1. On PE's main "Entry Options" page, click the Quick-Start link for SH3 Domain:Peptide Complex (proto-oncogene CRK, 1b07).
    2. Take a few minutes to digest the FirstView.
        Incidentally, this PDB file contains an unusual item: a one-residue chain (shown in FirstView as a small isolated ball). In actuality, this residue was part of a polyhistidine tag attached to the carboxy terminus of this domain, but recent PDB rules require these histidines to be assigned to a separate chain (even though they are covalently linked to the large chain A). Because of the his-tag is given a different chain designation than the remainder of chain A, chime shows (incorrectly) no backbone continuity, nor covalent bonds between chain A and the his tag.

      Salt Bridges:

    3. SELECT All
    4. DISPLAY Salt Br.
        Cation-anion pairs of protein sidechains close enough to form salt bridges are shown. The energetic significance of the cation-anion pairs varies depending on factors that PE cannot determine. Therefore these are putative salt bridges only. Note also that PE's salt bridge detection and display routine works only for protein, so omits possible salt bridges involving nonprotein moieties (such as DNA). (These could be included in the salt bridge options in Advanced Explorer.)

        Initially, the salt bridges are colored by chain. This makes it easy to spot interchain bridges, such as the one between the larger chain A and peptide chain C here.

    5. COLOR Element (CPK)
        Now the colors distinguish the anions from the cations.
        Advanced Explorer includes a form enabling ligand ions to be included in the salt bridge detection and display.

      Cation-Pi Interactions (still with 1b07):

    6. SELECT All
    7. DISPLAY Cation-Pi
      • Initially, the interacting cations and rings are colored by chain. This makes it easy to spot interchain bridges, such as the one between the larger chain A and peptide chain C here.
    8. COLOR Element (CPK)
        This makes it easier to distinguish Phe from Tyr.
        Advanced Explorer includes a form enabling ligand rings or cations to be included in the cation-pi detection and display.
     

  32. How can I measure distances and angles? Distances and angles specified by mouse clicks on atoms can be reported with the options displayed by DISPLAY Clicks. Optional: Try each of these options to see how they work.  

  33. How can I label an atom? An atom-labeling option is available via DISPLAY Clicks. Optional: Try labeling some atoms to see how this works.  

  34. How can I see the molecule in stereo? Rotating the molecule with the mouse (or with [Spin]) provides excellent 3D cues. Rotation conveys 3D structure effectively in class lectures, avoiding the need for cumbersome and expensive stereoscopic viewing methods (such as dual projectors and polaroid glasses).

    Individuals may find true stereoscopic viewing helpful for some kinds of images. This can be done without special equipment, but the ease with which it can be learned, and the visual fatigue which results, varies among individuals. My recommendation is that you give it a try, but if you find it too hard, don't worry about it. You can get along fine without it.

    Press the [Stereo] button. Now there is a split image which can be viewed in stereo. There are two kinds of split images: for convergent ("cross-eyed") or divergent ("wall-eyed") viewing. Convergent viewing is straightforward even with large images, such as on a computer screen. However divergent viewing becomes more difficult when the separation distance between images exceeds the interpupillary distance between your eyes. Therefore PE shows convergent stereo by default. Some people find one mode of viewing easier than the other. If you have difficulty achieving convergent stereo, or find it uncomfortable, try divergent.

    Convergent stereo viewing. (It is easiest if a friend reads this to you while you do it.) If you wear reading glasses, put them on. Turn off spinning. Press the [Stereo] button until the image is split. Position your head directly in front of the split image, not off to the side. Put your finger midway between the two images, near the top. Pick part of the image near the top that is easy to distinguish. Focus on your finger, and move it slowly towards your nose, keeping your focus on your finger. In the background, you should see the two images moving towards each other. The goal is to have them superimpose perfectly. If one is slightly higher than the other, tilt your head to the left or right until the alignment is perfect. Keeping your finger in focus, move it slowly towards or away from your nose until the images align perfectly. At that point, you should see the depth in the 3D view, and you can shift your attention away from your finger to the molecule. With practice, you can do this without using your finger, just crossing your eyes slightly until the images align.

    Divergent stereo viewing. First, click on Preferences below the message box, uncheck Stereo convergent, and click [Back]. Now click [Stereo] until the image is split again. Some people experienced with divergent viewing can align the images even when they are widely separated, but beginners should make sure the distance between images is slightly less than the distance between the pupils of your eyes (about two inches). The quickest way to reduce the separation is:

    (It is easiest if a friend reads this to you while you do it.) If you wear reading glasses, put them on. Turn off spinning. Put your nose between the two images, almost touching the computer screen. Don't worry about focus -- the image will be blurry, but you should see only ONE image. Pick a distinguishable reference point, such as the top of the image -- you want to see only one reference point, blurry but aligned. If you see two partially overlapping images, adjust the distance between images to be closer to the distance between your eyes. Once you see one (blurry) image with your nose almost touching the screen, move your head slowly away from the screen, keeping your eyes relaxed, gazing to infinity, with no effort to focus. The goal is to keep the central image aligned as you move away. Often you will need to tilt your head slightly to improve the alignment. As you move away, you should perceive three images -- the one in the middle is the aligned one. As you get far enough to focus clearly, you should see depth. With practice, some people can just gaze off to infinity and align the images (without starting close to the screen), and some can learn to do this even when the distance between images exceeds their interpupillary distance.

    More information on viewing stereo pairs, including those printed in journals.

 

Chapter IV
Finding and Saving the Molecule of Your Choice

     

  1. How do I find the molecule I want? In order to see a molecular structure in the Protein Explorer (PE), you must load an atomic coordinate data file for that structure. Such data files are commonly in the Protein Data Bank format, hence called PDB files.

    All published macromolecular structures are available from the Protein Data Bank, which has mirror websites around the world. So the PDB is the most comprehensive place to look for proteins, DNA, RNA, and polysaccharides. If the molecule you want is a popular one, you may find it most easily at PDB at a Glance, a subject categorized list. If your molecule is more esoteric, the best way to start searching the entire PDB dataset is with PDB Lite, a simple and clear search interface designed for nonspecialists who use the PDB infrequently. If you need a more advanced search, try the PDB's high-powered SearchFields (www.rcsb.org). Some searches can be done better with Jaim Prilusky's OCA (an enhanced version of what was offered by the former PDB when it was at Brookhaven National Laboratory). This link takes you to PDB Lite, where you'll see a link to OCA that also mentions cases that can be done better with one or the other searcher: OCA via PDB Lite.

    Protein Explorer can display any PDB file, not just those from the Protein Data Bank proper. PDB files can be obtained through the web for thousands of small organic molecules, for theoretical models (e.g. lipid bilayers and for noncovalent assemblies such as virus capsids. Several sources are listed at the Molecules Galore page of the Molecular Visualization Freeware site.

    For probable quaternary structures or "biomolecules", including virus capsids, search for the subunit module at the Protein Data Bank (see above). In PDB Lite "View/Analyze/Save" page, look near the bottom of the page for a link to Likely Quaternary Molecular Structure. In SearchFields, click on "Other Sources", then on the "MacroMolecule" link (if there is one -- not all structures have this link). For example, if you search for "poliovirus", the hits will include 2plv.pdb, and the Quaternary Structure link will offer a 31 megabyte file 2plv.mmol which includes all 60 subunits in the icosahedral capsid.

    Please note that all of the above links are available from PE's main Entry Options page -- so you don't have to come back here to find these sources of molecules.  

  2. How do I display my molecule in PE?

    1. PE's main Entry Options page lists a variety of methods. Start in the center gray box entitled Startup Options for Your Molecules.
    2. "Empty" PE. If you enter "empty" PE (see links on PE's FrontDoor), and then load a molecule within PE, in future sessions it will appear on the Select previously loaded PDB file menu at PE's "Load Molecules" control panel. This works both for local files and files fetched via Internet.
    3. As explained also on PE's main Entry Options page, if you know how to create an HTML file, you can create one which has links, each of which invoke PE with a prespecified molecule. The PDB files in each link can be either URL's or local files. The HTML file can be either on your local disk, or on a server. Here are Examples of Links to the Protein Explorer (PE) with Prespecified Molecules .

      Educators:
      Note that item C above is an excellent way to prepare a class home page which allows your students to see molecules you select.

    If you have no Internet connection, you can still explore molecules with PE. You will have to download and install PE and also download the PDB files of interest. You could download these items on a different computer which has an Internet connection, and transfer them to your computer via diskette, zip disk, CD, or other means.  

  3. How do I download a PDB file?

    There are a number of tricky details which can cause problems. Therefore, we strongly recommend that you use PDB Lite. It has detailed, click-by-click instructions specifically for Windows 95/98/NT or Windows 3.1 or Macintosh PPC. You get these instructions after you have found the molecule of interest, gone to the final screen ("View/Analyze/Save"), and clicked on the link Save xxxx.pdb. You can view this final screen directly:

    PDB Lite's Screen View/Analyze/Save 1HHO (oxyhemoglobin)
    Or you can view directly
    PDB Lite's detailed file-saving instructions.

    If you are already viewing the molecule in PE, you can save the PDB file directly from Chime:

    1. Click on the MDL frank to the lower right of the molecule to bring up Chime's menu.
    2. Select File, Save Molecule As.
    3. Change the filename to something descriptive of the molecule, or to xxxx.pdb (where "xxxx" is the PDB ID code). It is best if the filename ends in .pdb, and on Windows, is enclosed in double quotes. (This prevents Windows from tacking on unwanted file extensions).
    4. Change "Save In" to the desired folder/directory.
    5. Press "OK" to save the file.
    6. To verify, try loading the saved file with the [Browse] button on the Load Molecule page (select "New Molecule" in the PE Site Map).
     

    Chapter V
    Advanced Explorer

     
  4. Access to Advanced Explorer. Advanced Explorer can be reached in any of several ways:
    1. From QuickViews, via a link near the bottom of the menu frame, Advanced Explorer.
    2. By entering the command .x (period followed immediately by x). This works from any part of PE.
        (This is a command to PE, as distinct from a command to Chime. Click the question mark near the command entry slot for more information.)
    3. Automatically upon startup, after closing a session in which you checked the Expert Preference.
      • Preferences are accessed via a link beneath the message window.
      • Expert mode bypasses FirstView, and some help windows designed for novices no longer pop up.
    4. With a hyperlink, by including the query parameter x=1. This overrides the preference setting. Here are instructions.
     

  5. Advanced Explorer's Built-In Tutorials Several capabilities of Advanced Explorer have their own built-in tutorials. Although they rely largely on forms, filling in certain form elements requires some knowledge of Chime's command language terminology.  

  6. Chime's Command Language

    When you use the menus or buttons, PE sends commands to Chime. Frequent users of PE may wish to learn to enter commands directly. For some goals, directly entered commands are more efficient than the menus or buttons, and some results can be acheived only with manually entered commands.

    Groups of commands sent to Chime in a single package are called command scripts, or just scripts. Most of the menus and buttons in PE display their scripts in PE's message window. One exception is the FirstView script that creates the first image you see after loading a new molecule. The FirstView script can be messaged with an option accessed with the [Message Control] button near the message box. In QuickViews, the most complex DISPLAY scripts are not messaged (Cation-pi, Salt Br.). However, a link in QuickViews middle help window will display these scripts.

    The easiest way to begin learning the command language is by watching the messages generated by PE's buttons and menus. Then try entering these commands (or variations on them) in the slot above the message window, and observing what they do to the image. Be aware that on Windows only, messages appear in reverse order, newest at the top. This avoids having the newest messages always out of view. (The order can be changed in the Preferences. On Macintosh, Netscape's form box is intelligent enough to scroll to the bottom automatically when new text is added, so the message order defaults to newest at the bottom.)

    When entering commands, PE's command aliases can save a lot of typing. For example, typing "s bb" is expanded automatically to "select backbone". To view a complete list of aliases, click on the Aliases link below the message box. As explained there, it is easy to add, delete, or modify aliases to suit your preferences.

    In addition to the extensive command language understood by Chime, there are a few commands understood by Protein Explorer (intercepted and not forwarded to Chime). The most useful: typing a comma as the first character in the command slot immediately recalls the previous command (without pressing Enter). More information, and a complete list of these can be displayed by pressing the blue question mark near the command entry slot.

    Here are some sources of a more systematic introduction to the command language.

    Here are some tricky issues to be aware of concerning the command language.

    Chime's Menu. Clicking on the MDL frank below the molecule image, at the bottom right corner, opens Chime's menu. This is a powerful menu worth getting familiar with. Many of its actions can be done better in QuickViews because they are better organized in those menus, and are accompanied by help and color keys. However, in the Select branch of Chime's menus you can see most of the predefined terms that Chime understands. This is a handy place to look up terms you may need to complete a manually entered select command.

    Note that Chime's Select menu messages its commands; but all the other Chime menu branches don't (such as Display, Color).

    Command scripts can be saved into plain text files (.txt, ASCII or DOS format), then played back later. Script filenames should always end in ".spt" (conventionally mapped to MIME type application/x-spt). disk files and run in PE. In order to run them from your local disk, you must download PE and set a project folder (see the Project Folder link beneath the message window).

    Chime-saved scripts. Chime can automatically generate a script that will produce the image displayed. Click on MDL (lower right corner of Chime) and select Edit, Copy Chime Script. Then paste the contents of the clipboard into a text editor (Windows: Wordpad or Word; Macintosh: BBEdit or Word), being sure to save it as plain text. Chime-saved scripts tend to be unnecessarily long and may take an unnecessarily long time to produce the image -- see the method for Shortening Scripts Saved from RasMol or Chime. For more information on creating web-deliverable tutorials with your scripts, see Presenting RasMol-Saved Scripts in Chime.

     

  7. Multiple-model ensembles (NMR).
    Initial view of 4-model NMR file 1abt.pdb, containing disulfide bonds.

    Multiple models typically occur in PDB files resulting from NMR studies. They may also occur in PDB files designed to show conformational changes (morphs), or structural alignments of two or more molecules. Your first clue that you are looking at a multiple-model ensemble depends on whether PE is in expert mode or not.

    In either mode, when you go to Advanced Explorer, a special option will appear at the top of the menu, NMR Model Selection.

    Let's try some examples:

    1. If you're on a modem, start a session for the tiny NMR file 1tor. If you have a fast connection, try 1abt. (These examples have disulfide bonds; see below.)

    2. The number of disulfide bridges reported by "show info" is incorrect (much too high). There is a bug which fails to distinguish models in assigning bridges, so nonexistent bridges are assigned between sulfurs proximal in space but in different models. The best way to count bridges is in QuickViews, with DISPLAY SSBonds. This shows only the first model. You can then count the SSBonds by careful inspection while rotating the molecule with the mouse.
    3. For multiple model files, the number of chains per model is the "Number of Chains" divided by the "Number of Models" from the "show counts" report (accessed in the Molecule Information Window). For multiple-model files, the command color chain gives each chain in each model a different color -- usually not very useful!
    4. Go to Advanced Explorer and enter the NMR Model Selection control panel. When you enter this, if you are not in expert mode, it will offer to "Apply N->C Rainbow (Group) Colors" -- accept the offer. (If you are in expert mode, this is the initial view so the offer is not made.) You restore this view at any time by pressing the [All] button.

      Although there is no tutorial here, there is extensive documentation. Be sure to press all the blue question marks and read the help! Notice the multiple, thin backbone traces. When the initial view appears, notice the report "Number of Models" in the message window (it doesn't appear at all for 1-model files).

    5. NMR ensembles show flexibility of certain parts of a molecule. Because of the flexibility, the models will not be closely aligned in these regions. These regions are commonly the "loose" ends of chains, often long loops on the surface with no secondary structure and sometimes chains connecting two compact and stable domains. The carboxy terminus of 1abt shows multiple conformations, as do the two surface loops containing residues 35 and 50. A more extreme loose end is seen in calcium-free calmodulin, 1cfc (4.7 megabytes). A great example of a loose end which is myristoylated is seen in 1jsa (caution! 6 megabytes). The Protein Morpher animates flexiblity of the interdomain linker in calmodulin.

    6. The [Auto] button on the NMR Model Selection page animates these sorts of conformers, simulating thermal motion. Try it!

    Brief introductions to NMR methods for determining macromolecular structures are in the Nature of 3D Structural Data overview at the PDB, many biochemistry textbooks, and Branden & Tooze. Unlike X-ray crystallography, NMR methods yield an ensemble of models, all consistent with the experimental data. At the PDB can be found data files containing anywhere from two (1hpn) to more than 40 models (1yuj: careful, more than 6 megabytes!). The authors of these data files have made a judgement as to how many models to provide. Sometimes the authors also deposit a PDB file containing one model which is an energy-minimized average of the ensemble (1cfd [191 kilobytes] is an average of 1cfc [4.7 megabytes]).

    Occasionally multiple models will be published as the result of X-ray diffraction studies (1cm4 [451 kb]). More commonly, sidechains of certain residues may be given multiple positions (R134, Y192, P195, R207, L216, R219 in 1lkk [340 kb]), perhaps because of evidence for multiple conformations. Because Chime assigns bonds dynamically based on interatomic distances, and because typically these sidechain conformers are not designated as separate models in the PDB file, Chime creates "nests" of inappropriate bonds in these areas.

     

  8. Further self-guided exploration. Now you're on your own. Apply the methods in the previous sections of this tutorial to new molecules of interest. Specialized knowledge of the properties of your molecule will help, so read the relevant literature!

Notes  

Illegal atoms in PDB files. The PDB format requires that every atom which is not a member of one of the standard 20 amino acids or 5 nucleotides be designated as a hetero atom (HETATM record). PDB-format files obtained from sources other than the Protein Data Bank proper sometimes contain "illegal" atoms which should be designated HETATM but are instead designated ATOM. To ensure that such atoms are obvious, they are rendered in ball and stick in PE's initial view (see shared\view1.spt, shared\view1nmr.spt).

One-residue chains. There are over 90 cases of PDB files containing one-residue chains (e.g. 4csm, 4jdw, 1arj, 1rnm). Since there is no backbone trace for a single residue, the alpha carbons of amino acids and phosphorus atoms of nucleic acids are shown as small spheres in the initial view offered by PE. This guarantees that the rare one-residue chain will not be completely invisible. (An example with two 2-residue chains is 1dn8.)  

"slab" -- a misnomer? The term "slab" means a slice with thickness. Many molecular graphics programs, such as Mage, have a true slab mode in which both the portions in front of, and behind, the slab are hidden. Since in RasMol & Chime there is only one slicing plane, instead of the two needed to make a true slab, the term "slab" is somewhat of a misnomer in these programs. (See set slabmode for some interesting variations which are available.)

END OF PROTEIN EXPLORER TUTORIAL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  use of chime's menu to look at range of residues. 1qmg has 4 heteros and all 20 aa's. 1d66 lacks GMF. NB residues < 3 chars absent, e.g. CD, A T G C.