RasMol for Dummies

Chapter I
A Tutorial for the Rasmol Basics

1. How to get protein/DNA 3D structure file from Protein Data Bank?
   • See " Getting Protein structure " section
2. How many chains are there?

   Now type the following commands in the Command Line window.

    

   Command:

   reset
   rotate z 90
   zoom 150
   rotate y 40
   backbone (on)
   color chain

   Click on each chain to report its ID letter code (last item of the report).
   
   show sequence

   This command reports complete amino acid sequence and information about chains.

3. Is there anything else in this PDB file besides the protein/DNA chains?

   Command:

   select hetero
   Select things like water, metal ion or anything else except proteins, RNA and DNA.
   spacefill
   color cpk
   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.
   

    

   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

    

4. Where are the hydrophobic amino acids?

   Command:

   select hydrophobic
   color magenta
   wireframe 0.4

   Note amphipathicity of alpha helices.
   

   select not water
   spacefill (on)
   slab (on)
   Slice thru the molecule to look at distribution of hydrophobic and hydrophilic residues.
   Move the slab plane with the mouse.

    

    

5. What holds the Cd ions in place?

   Commands:

   slab off
   select all
   backbone 0.3
   color chain
   select cd
   spacefill (on)
   color cpk
   select within(2.6, cd)
   This selects all atoms within 2.6 Angstroms of the Cd++ ions.
   spacefill on
   color cpk
   Click to discover the identity of the caging atoms.
   

    

6. How do we save this view?

   commands:

   save script gal4.spt
   zap

    

7. How do we restore the view later?

   Commands:

   script gal4.spt

    

8. Where are the alpha helices and beta strands?

   Commands:

   Select all
   Backbone (0.3)
   color structure

   This colors alpha helices purple, and beta strands yellow (there aren't any beta strands in 1d66.pdb).
   
   structure
   color structure.

   This forces RasMol to make its own determination, i.e., using the DSSP definition of secondary structure.
   Notice the appearance of blue "turns".
   

    

9. How do I find the distance between two atoms?

   Commands:

   spacefill (on)
   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
   The number disappears!
   monitor off
   The dotted line disapperas!
   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

    

10. How do I find the bonds between protein and DNA?

    Commands:

    reset
    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.
    color cpk
    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).
    

     

11. How do I see the inside of a molecule?

    Commands:

    Don't rotate the molecule with the mouse at any time during this sequence.
    reset
    select All
    spacefill
    color Chain.
    rotate x 83
    zoom 200
    set hetero off
    Toggle off waters
    select dna
    color cpk
    slab
    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.)

12. How do I keep the DNA from rotating off screen?

    Commands:

    reset
    restrict none
    Clear the screen
    restrict dna
    spacefill
    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.

     

13. How do I get multiple representations of the same atoms?

    Commands:

    restrict :d
    color cpk
    :d means all atoms in chain D.
    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

     

14. How do I label an atom?

    Commands:

    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 %n%r
    Label residue name and number
    label
    Label all information of the selected atoms
    label off

    Notes:

    The options for "set picking" command are:

    ident	report an atom identity
    distance	report the distance between two atoms
    angle	report the bending angle defined by three atoms
    torsion	report the torsional angle defined by four atoms
    monitor	Toggle the displayt of the measurement such as distance, bending or torsional angles in the Main window
    label	Toggle the display of an atom label on a given atom
    center	set the atom picked as the center of rotation

    The available label specifiers are

    %a	Atom Name
    %n	Residue Name
    %r	Residue Number
    %c	Chain Identifier
    %i	Atom Serial Number
    %e	Element Atomic Symbol

     

     

15. How do I see the molecule in stereo?

    Commands:

    stereo (on)

    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 macweb.acs.usm.maine.edu/chemistry/GR/GraphicsGallery/StereoView.html

     

16. How to specify chains, residues and atoms of your molecule?

    The most general syntax of specifying an atom in Rasmol is, for example,

    lys45:a.nz
    This specifies N zeta of lysine 45 of chain A.

    Some other examples of the atom expressions are:

    :a	All atoms in chain A
    lys:a	All glutamate in chain A
    45:a	The 45th residue in chain A
    *45:a.nz	The N zeta atom of the 45th residue in chain A (Note that the asterisk is necessary, if the residue name is not explicitly specified.)

17. Useful tips

    To erase the display of molecules in the Main Window, type

    restrict none

Chapter II
Exploring the Molecule of Your Choice

Getting Started

Now fetch a PDB file for the molecule of your choice from pdb.life.nthu.edu.tw. (See Chapter 1)

1. How many chains are there?

   Commands:

   Backbone
   color 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,
   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?

   Commands:

   select hetero
   spacefill
   color 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?

   Commands:

   cartoon
   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?

   Commands:

   color 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,
   set hetero off (leaving Hetero Atoms unchecked), then again type
   color 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?

   Commands:

   select all
   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?

   Commands:

   wireframe;
   ssbonds 0.8

   The disulfide bonds should now be visible as rods 0.8 Angstroms in radius. It may help to
   color ssbond(s) 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:
   Select All
   and repeat the above commands.
   Backbone
   color 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 ssbond(s) backbone.

    

7. Where are the hydrogen bonds?

   Commands

   select All
   backbone
   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).