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

Overview.

• Beginners should first read RasMol Quick Start at www.umass.edu/microbio/rasmol/rasquick.htm . There you can get RasMol and the 1d66.pdb atomic coordinate file needed for the first chapter below.
• For background, get the short, excellent and readable Introduction to Protein Structure by Carl Branden and John Tooze, Garland Publishing, 1991 (www.garlandpub.com).
• This document has two chapters. In the first, you apply specific commands to a specific molecule, 1d66.pdb, a protein:DNA complex. In the second, general instructions are listed which are applicable to any molecule of your choice.
• Other tutorials on RasMol are at www.umass.edu/microbio/rasmol/softhelp.htm . The RasMol Reference Manual, answers to Frequently Asked Questions (FAQ), and other helpful documents are at www.umass.edu/microbio/rasmol/getras.htm#rasmanual

Getting Started

• Commands below preceded by M are best done from the pull-down Menus. Commands not preceded by M must be typed in the white command-line window. (RasMol has two windows, one black and one white. On Windows, the white command-line window starts minimized. Look for it on the taskbar. On Macintosh it starts behind the black window. Move the white window below the black window, but keep the bottom line in view.) Commands listed below separated by semicolons should be typed on separate lines into the white window, pressing Enter after each command. Comments are italics -- these are not commands.
• Run RasMol, and do M(enu) File -> Open. Select 1d66.pdb (gal4 transcriptional regulator complexed to DNA) (If you don't see 1d66.pdb, detailed troubleshooting is available at www.umass.edu/microbio/chime/prsswc/prssft.htm ).
• After a select command, subsequent commands affect only the selected atoms. The display representations of unselected atoms will remain unchanged. A restrict command is like a select command except that it hides all unselected atoms.

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

7. 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".

8. 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

9. 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.

   dots

   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).

10. 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

11. 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.

12. 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

13. 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

14. 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

• Obtain a PDB file for the molecule of your choice. See Molecules Galore at www.umass.edu/microbio/rasmol/whereget.htm
• Look in the PDB file (use a text viewer such as Wordpad or Word) for references to journal articles. Reading these papers is often essential to understanding what you are looking at! For example, how much of the intact molecule is represented in the PDB file? How was it prepared?
• Use RasMol's File, Open menu to display your molecule. If you don't see your molecule, detailed troubleshooting is available at www.umass.edu/microbio/chime/prsswc/prssft.htm
• If a command has no effect, you may have nothing selected to which it applies. For example, if you select hetero and later color structure, there are no proteins selected to which the secondary structure color scheme applies, so nothing happens. Solution: select all, or select the relevant moieties and re-issue the command.

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