What can you learn about a protein/DNA molecule with RasMol?

by Eric Martz, 6/97 (revised 8/6/97, 9/14/97, 2/27/98, 4/3/98, 4/27/98, 10/26/98)
If you are reading a paper version of this document, it is also available at http://www.umass.edu/microbio/rasmol/raswhat.htm


Getting Started

Chaper I
Questions & Answers about 1d66.pdb

  1. How many chains are there?
    reset; rotate z 90; zoom 150; rotate y 40
    Type each semicolon-separated command on a separate line, pressing Enter after each one.
    This sequence positions the molecule nicely.
    M(enu) Display -> Backbone, M Colours -> Chain
    Now each chain is a different color.
    Click on each chain to report its ID letter code (last item in the report).

  2. Is there anything else in this PDB file besides the protein/DNA chains?
    select hetero; M Display -> Spacefill
    Now you see oxygens from water in the X-rayed crystal.
    M Colours -> CPK
    CPK is the Corey-Pauling-Koltun color scheme.
    Click on an atom to find out what element its color signifies.
    restrict not water
    This hides water; click on what remains to find out what it is. The older PDB standard and files have some ambiguities; CD could mean either carbon delta or cadmium -- here it is the latter. The physiologic metal for gal4 is zinc; cadmium was substituted in the crystallized protein.

    For a general introduction to how to select or restrict (hide) atoms, residues, chains, ligands, groups of residues (such as hydrophobic or charged) and nearest neighbors, see Select Commands in Chime and RasMol (www.umass.edu/microbio/rasmol/seleccmd.htm).

    Mouse Click and Drag Summary
    Action Windows Macintosh
    Rotate X,Y Left button Unmodified
    Translate X,Y Right button Command
    Rotate Z Shift-Right Shift-Command
    Zoom Shift-Left Shift
    Slab Plane Control-Left Control

  3. Where are the hydrophobic amino acids?
    select hydrophobic; color magenta; wireframe 0.4
    Note amphipathicity of alpha helices.
    select not water; M Display -> Spacefill; M Options -> Slab mode
    Slice thru the molecule to look at distribution of hydrophobic and hydrophilic residues.
    Move the slab plane with the mouse.

  4. What holds the Cd ions in place?
    M Options -> Slab mode Toggle the slab mode off.
    M Edit -> Select All
    M Display -> Backbone; M Colours -> Chain
    select cd; M Display -> Spacefill; M Colours -> CPK
    select within(2.6, cd)
    This selects all atoms within 2.6 Angstroms of the Cd++ ions.
    M Display -> Spacefill; M Colours -> CPK
    Click to discover the identity of the caging atoms.

  5. How do we save this view?
    save script myview1.spt; M File -> Close

  6. How do we restore the view later?
    script myview1.spt

Your RasMol-saved views (scripts) can be played back smoothly in the correct order from a master script. See Preparing RasMol-Saved Scripts for Teaching (complete with troubleshooting) at www.umass.edu/microbio/chime/prsswc/prssft.htm.

Your RasMol-saved scripts can easily be installed in web pages for delivery via Chime (no programming needed!). See Presenting RasMol-Saved Scripts in Chime where you can download the template at www.umass.edu/microbio/chime/prsswc/template.htm.

  1. Where are the alpha helices and beta strands?
    M Edit -> Select all; M Display -> Backbone; M Colours -> Structure
    This colors alpha helices purple, and beta strands yellow (there aren't any beta strands in 1d66.pdb).
    structure; M Colours -> Structure.
    This forces RasMol to make its own determination.
    Notice the appearance of blue "turns".

  2. How do I find the distance between two atoms?
    M Display -> Spacefill; set picking distance
    Now click on two atoms, and watch the report in the command line window.
    If you want label the two atoms with the distance, try this:
    set picking monitor
    Now click on two atoms near the edge of the molecule.
    (This will work best if there is black background between the two atoms.)
    Watch what these do:
    color monitor white; set monitor off; monitor off
    set picking ident
    The above restores the normal clicking function of identifying the atom.
    RasMol can also report angles and torsion angles. See www.umass.edu/microbio/rasmol/distrib/rasman.htm#setpicking

  3. How do I find the bonds between protein and DNA?
    reset; M Display -> Backbone;
    color green; backbone 0; rotate z 91; translate y -17; zoom 200
    select dna; color white; spacefill; center selected
    select dna and backbone; color yellow
    Now you can see the DNA, with backbone and base pairs in different colors, and you can see the backbone of the protein as a thin green line.
    select within(3.1, dna) and not dna
    If you left out "and not dna", you'd select the DNA also! The above command should select 35 atoms.
    Now press the "up arrow" key until you see the "within" command, and add to the end of it and not water. Press Enter, and 19 atoms should be selected.
    spacefill 0.6;
    Now the putatively bonded protein atoms are small solid spheres within dot-spheres, while the hydrogen-bonded water oxygens are hollow red dot-spheres.
    select within(3.1, protein) and dna; color cpk
    Now you can zoom in and click on prospective donor and acceptor atoms to identify the residue to which they belong and evaluate the liklihood that a given pair is in fact hydrogen-bonded (or otherwise bonded).

  4. How do I see the inside of a molecule?
    Don't rotate the molecule with the mouse at any time during this sequence.
    reset; M Edit -> Select All;
    M Display -> Spacefill; M Colour -> Chain.
    rotate x 83; zoom 200
    M Options -> Hetero Atoms (Toggle off waters)
    select dna; color cpk;
    M Options -> Slab Mode (Toggle on slab mode)
    The front half of the molecule has been cut away. You see the cut face, and everything behind it.
    set slabmode section;
    Now only the cut face is show. Everything in front of and behind the cut plane is hidden. Only the atoms hit by the "knife" are shown.
    slab 76
    Now you see a GC base pair, cut through the plane where the three Watson-Crick hydrogen bonds are. (This won't work if you moved the molecule with the mouse anytime since the reset.)
    slab 68
    What is this? Use the mouse to move the slab plane (Hold down Ctrl, then click and drag up and down). Can you find a base pair which is completely out of Watson-Crick position? (Answer is at the end of this document.)

Controlling RasMol's Display

  1. How do I keep the DNA from rotating off screen?
    reset; restrict dna; rotate z 90; zoom 200
    Try rotating around the axis of the DNA helix (move the mouse up and down).
    Notice how the DNA rises and falls as it rotates around the center of mass, which includes the invisible protein.
    center selected
    Now try again and notice the difference.

  2. How do I get multiple representations of the same atoms?
    restrict :d; M Colours -> CPK
    :d means all atoms in chain D.
    M Display -> Backbone, M Display -> Ball & Stick
    Notice how you get one or the other but not both when you use the Display menu. This is because each representation on the Display Menu turns off all other representations for the selected atoms. In contrast, when representations are applied from the command line, they do not turn off other representations.
    backbone 1.
    Be sure to include the decimal point after the one, which makes RasMol interpret it as Angstroms.
    When you type display commands, existing representations are not turned off (unlike with the display menu).
    "Sticks" are wireframe with a nonzero radius. Balls are spacefill with a uniform radius. Watch these:
    spacefill off; wireframe 0.5; wireframe 0.1; spacefill 0.3; backbone 0.1; zoom 500

  3. How do I label an atom?
    set picking label
    Now click on a few atoms.
    color labels white; label off; set picking ident
    Click on an atom and notice its atom ID number (3rd word in the report). We'll refer to the number as ### in the command below.
    select atomno = ###; label "My Favorite Atom"
    label off

  4. How do I see the molecule in stereo?
    M Options -> Stereo
    Now you need to translate to the left to center the image.
    By default, you get cross-eyed stereo. To get wall-eyed stereo:
    stereo -5
    Viewing stereo takes practice, can be hard on the eyes, and is not necessary for most purposes. Rotation without stereo gives you an excellent perception of major 3D relationships. However, if you view molecular graphics frequently, learning how to view images in stereo will enable you to see complex spatial relationships more clearly. Gale Rhodes has provided an excellent introduction to stereo viewing at www.usm.maine.edu/~rhodes/0Help/StereoView.html

Chapter II
Exploring the Molecule of Your Choice

Getting Started

  1. How many chains are there?
    M Display -> Backbone; M Colour -> Chain
    At any time, you can restrict your view to one or a subset of the chains present. Click to find out the chain letter. Suppose you want to hide all chains except B and D: restrict :b or :d . To restore the view to all chains, M Edit, Select all.

    If you want to look at only part of a large PDB file (greater than 500,000 bytes), it will greatly improve RasMol's performance if you make a copy of the PDB file from which you delete all atoms except the series you wish to view. To do this, select the desired atoms/chains/residues/ligands in RasMol, then save pdb filename.pdb Open the new PDB file in RasMol for further work. Be sure you didn't omit important ligands!

  2. Are any ligands present?
    select hetero; M Display -> Spacefill; M Colour -> CPK
    Click on a ligand to see its 3-letter "residue" code, assigned in the PDB file. You can select ligands with their 3-letter codes. Often the PDB file contains remarks about the ligands (open it in a text viewer, such as Wordpad or Word).

    Often the view is cluttered with water oxygens. (Remember, hydrogens cannot be resolved by X-ray crystallography.) Protein crystals are quite "wet" and gelatinous; the structures obtained from crystals agree well with structures obtained from proteins in solution by NMR. Most of the water molecules in crystals diffuse randomly, making them "blurry" and invisible. The rare visible water molecules were tightly bound and immobilized. To hide the water, restrict not water .

  3. What is the secondary structure?
    M Display -> Cartoon; M Colour -> Structure
    Alpha helices are red, beta strands yellow, turns blue, and everything else is white. Often the PDB file specifies secondary structure with HELIX and SHEET records. If it does, RasMol obeys it. If it does not, RasMol makes its own determination. You can force RasMol to make its own determination with the command structure.

  4. Where are the N and C termini?
    M Colour -> Group (Backbone display is best for this.)
    Each chain should begin blue, changing color through a rainbow series (green, yellow, orange) and end in red. If the chain(s) is mostly blue, M Options -> Hetero atoms (leaving Hetero Atoms unchecked), then again M Colour -> Group.

    Here are mnemonics. Synthesis begins with the old end; new residues are added to the new end.

    • Blue = cold = old (N terminus of proteins, 5' end of nucleic acids)
    • Red = hot = new (C terminus of proteins, 3' end of nucleic acids)
    • The amino terminus has the blue CPK color of N; the carboxy terminus, the red CPK color of O. The 3' hydroxy terminus of nucleic acids has the red CPK color of O.

  5. Where are the hydrophobic side chains?
    M Edit -> Select All; M Display -> Spacefill
    select protein; color [180,180,180]
    select protein and backbone; color [100,0,100]
    select protein and not (backbone or hydrophobic); color magenta
    Optionally select not protein; color greenblue
    The hydrogen bonding requirements of backbone atoms are generally satisfied within the backbone. Hence backbone atoms are not usually extensively hydrogen bonded to nonbackbone atoms. Backbone atoms are assigned a dark (magenta) color, indicating they are weakly hydrophilic. Hydrophobic side chains are gray to indicate their high carbon content. Polar or charged sidechains are bright magenta to indicate their strongly hydrophilic nature. Magenta is used to represent a mixture of equal parts of red and blue (red for O=positive and blue for N=negative charges or partial charges).

    Large patches of hydrophobic sidechains on the surface of the protein suggest that these regions contact something hydrophobic, rather than water. Examples could be proteins that form multimers with each other, or proteins that surround themselves with or bind to lipids.

  6. Where are the disulfide bonds?
    M Display -> Wireframe; ssbonds 0.8
    The disulfide bonds should now be visible as rods 0.8 Angstroms in radius. It may help to color ssbonds yellow. The first ssbonds command reports the count as "Number of bridges"; if the count is zero, your molecule doesn't have any! If you got a nonzero count, but don't see any ssbonds, you probably didn't have the cystine-containing protein selected. Solution: M Edit -> Select All and repeat the above commands.
    M Display -> Backbone; M Colour -> Chain
    Now only the alpha carbon positions are shown. None of the other atoms in the cystines are shown. Since the ssbonds connect sulfur atoms on the sidechains of cystines (not shown), the ssbonds appear to "float in space". You can render the ssbonds as connecting the alpha carbons of the cystines with set ssbonds backbone.

  7. Where are the hydrogen bonds?
    M Edit -> Select All; M Display -> Backbone; M Colour -> Structure
    restrict helix; backbone 0; hbonds 0.5; color hbonds white
    RasMol shows only the backbone hbonds. It is not capable of displaying hbonds between sidechains, beween chains, between ligands and their binding sites, etc.

    As with the ssbonds, the hbonds appear to be "floating in space" since the atoms which they bond are not shown in a backbone display. The hbonds can be schematized as linking backbone alpha carbons with:

    set hbonds backbone
    Now hbonds off, and repeat the above sequence but instead of restrict helix, substitute restrict sheet. And again, substituting restrict not (helix or sheet).

  8. Where are the interchain bonds? The ligand:protein bonds?
    As explained under hydrogen bonds, the hbonds command in RasMol is not capable of displaying interchain hbonds. To find bonds of all sorts between moieties, you must use the within command, customizing the example in the section above on 1d66.pdb to your molecule. A standard hydrogen bond has a length of 3.0 Angstroms between the donor and acceptor atoms (for example, N and O). This is made up of a 1.0 Angstrom covalent bond between the hydrogen and its covalently bonded atom, plus a 2.0 Angstrom hydrogen bond between the hydrogen and the hbonded atom. Therefore, a distance of 3.0 or generously, 3.2 Angstroms is appropriate for the within command. Hydrophobic bonds tend to be longer (carbon to carbon), up to 4.0 Angstroms.

    There is no ideal way to "paint in" an arbitrary hydrogen bond. The best way to indicate such a bond is with the set picking monitor command (see section above entitled "How do I find the distance between two atoms?"). This draws a dotted line between the atoms, optionally labeled with the distance in Angstroms. The limitation is that you cannot make the monitor line thick (as in a stick representation of a bond).

    The above methods using the within command are quite laborius. Therefore an automated interface has been developed called the Noncovalent Bond Finder at www.umass.edu/microbio/chime/find-ncb/index.htm

Answer to base pair question in item 10: slab 53. Inquiry from the author of the paper (Ronen Marmorstein) revealed that the position of cytosine 28 is in error. There is no reason to believe that this base would be pulled out of Watson-Crick position by some unusual interactions with neighboring moieties. The lesson is: don't believe everything in a PDB file. Just because every atom is assigned a precise position doesn't mean the positions are correct!

Feedback to emartz@microbio.umass.edu .