INDEX     BCH441 COURSE     HTML TUTORIAL     BIOINFORMATICS A RasMol tutorial

Outline Welcome to a short tutorial on RasMol. Molecular Modeling RasMol Installation Downloading Coordinate Files Stereo Vision Representation Selection Color H-Bonds Measurements Surfaces and Slabs Saving and Printing Scripts Web Resources Feedback

Molecular Modeling Structure is a three-dimensional concept and it does not follow readily from any linear or tabular description of the protein. To study structure, it is necessary to develop an intuitive feeling of the spatial arrangement of typical protein features and this ultimately requires to experience proteins in 3-D. Roger Sayle's program RasMol is one of the most widely distributed programs for molecular modelling and viewing of small molecules, proteins and nucleic acids. It is compact, fast, versatile, rich with essential features and free. It is a command line driven program and can be scripted and the source code is available. For taking a quick look at things, or for teaching purposes, it is unsurpassed. That is not to say that there are not alternatives (see ...  for an annotated list of available visualization software). Unfortunately, the most useful alternative to RasMol for teaching purposes, CHIME , a web-browser plugin, only works with specific versions of IE, since the support for plugin interfaces was unexpectedly discontinued by Microsoft in the summer of 2001. MDL has since worked on updates, but CHIME will not run with IE 6. For the research communities, this is a great loss and there are still no good alternatives available to use with Web pages. Rather than expect users to run a specific constellation of browsers and plugins, we will use short scripts in our Web pages, which can be copied and pasted into the command-line window of RasMol. Molecular Graphics Here are some examples for molecular graphics: an imunoglobulin FV fragment rendered with MOLSCRIPT, a Pleckstrin Homology domain rendered with Raster 3-D, and GFP, ray traced with POV-ray, with a glowing fluorophore in the centre. While rendering quality of such images is usually superior to a simple molecular viewer, such as RasMol, static images are of course not interactive.

RasMol Installation Instructions for downloading and installing a current version of RasMol can be found at the OpenRasMol.org  Website. LINUX, Mac and many other binaries (even Windows) are available. The latest stable release is 2.7.1.1. Note that Microsoft Windows users need to install RasTop since Windows versions of Rasmol do not have copy and paste functionality. You may consider configuring RasMol as a helper application for your Web browser. See the information page on Configuring Netscape ... at the University of Massachsetts RasMol site. When you start the program, it will open two windows: a command line window and a viewer window. Type help into the command line window for a brief overview. Not all commands that work in this release are documented in the help feature. Here are three other sources of help (besides the help-file that came with your download:      Quick Reference A PDF for a quick reference to important commands.      RasMol Manual The online manual for RasMol 2.7.1.1      Search ... Since RasMol is so widely distributed, using a Web-search engine is quite an effective way to get examples for the correct use of specific commands or to find how to solve certain non-obvious problems.

Downloading coordinate files 3-D molecular coordinate files for proteins and (some) nucleic acids are held at the Protein Data Bank (PDB) . The following links will download coordinates to your disk:    2IMM Immunoglobulin VL: Variable Domain of the Light Chain (114 residues, 2.0 Å resolution).    1A6M Sperm-Whale Oxymyoglobin (151 residues, 1.0 Å resolution).

Stereo Vision Being able to visualize and experience strucutre in 3-D is an essential skill, if you are at all serious about understanding the molecules of molecular biology. Even though hardware devices exist that help in the three-dimensional perception of computer graphics images, for the serious structural biologist there is really no alternative to being able to fuse stereo pair images by looking at them. RasMol is an excellent tool to practice stereo vision and develop the skill. Stereo images consist of a left-eye and a right-eye view of the same object, with a slight rotation around the vertical axis (about 5 degrees). Your brain can accurately calculate depth from these two images, if they are presented to the right and left eye separately. This means you need to look at the two images and then fuse them into a single image - this happens when the left eye looks directly at the left image and the right eye at the right image. In fact, it can also be the other way around and some people find convergent (cross-eyed) stereo viewing easier. I recommend the divergent (wall-eyed) viewing - not only because it is much more comfortable in my experience, but also because it is the default way in which stereo images in books and manuscripts are presented. In order to visually fuse stereo image pairs, you need to override an ocular reflex that couples divergence and focussing, this is something that needs to be practiced for a while. Usually 5 to 10 minutes of practice twice daily for a week should be quite sufficient. It is not as hard as learning to ride a bicycle, but you need to practice regularily for some time, maybe 10 or 20 sessions of 3 to 5 minute over a period of a week or two. Once you have acquired the skill, it is really very comfortable and can be done effortlessly and for extended periods. You will enter a new world of molecular wonders ! Here are step by step instructions of how to practice stereo-viewing with RasMol. Load a small protein into RasMol and display this as a simple backbone model. Type set stereo -5 in the command line (the RasMol default is cross-eyed, thus the need to specify a negative rotation angle). Resize the window, until two equivalent points on the protein are the same distance on the screen, as your eyes are apart (this is usually about 6 cm). Touch your nose to the screen and look at the two images. They will be blurred and out of focus, but should appear as a three-dimensional object. Slowly rotating the protein helps. Once you see the object in 3-D, try to move your head backwards slowly, until the structure comes into focus by itself. Do not voluntarily try to focus, since this will induce your eyes to converge and you will lose the 3-D effect. When you lose the 3-D effect, start over. Practice this patiently, two times daily for some 3 to 5 minutes. Stop, when your head feels funny. Don't force yourself. It should take you about a week to master this, with regular training it will become very easy. And, the best thing is, you do not easily forget this skill. It is like riding a bicycle, equalizing pressure in your eustachian tubes while scuba diving, or circular breathing to play the didgeridoo: once you teach your body what to do, it remembers. And expands your horizon. Below, there is a script to set up a stereo-view of 2IMM. Simply copy the script from the textarea and paste it into the RasMol command line window. The commands get executed line by line and it is easy to change parameters and arguments and see what effect this has. reset select all wireframe off center rotate z 133 rotate y 24 rotate x 42 backbone 150 color [100,100,100] select 27-32, 49-55, 89-98 color [3,252,182] select all set stereo -5 set background [170,170,255] A set of script commands for a simple view of 2IMM, suitable to practice stereo viewing. 2IMM in stereo This is approximately the result of the above commands in RasMol. The domain is shown as a backbone tube, connecting C-alpha atoms, the CDR regions are highlighted in light-green. Here are some suggestions for stereo viewing mini-projects, so your practice sessions do not become monotonous. They are ordered from the most simple scenes to progressively more complicated molecules. 3D mini-project topics PDB source files Study individual amino acids and memorize the spatial arrangement of the groups around the chiral centre (the C-alpha atom) in this L-amino acid. Alanine Cysteine Aspartate Glutamate Phenylalanine Glycine Histidine Isoleucine Lysine Leucine Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine Study (and remember) the correct chirality of the Threonine side-chain. Threonine Download and study small molecule cofactors from HIC-UP. Some samples are linked here. ATP Arachidonic acid Beta-carotene Biotin Caffeine FAD FMN Phycoerythrobilin Testosterone Study an alpha-helix. Concentrate on how the carbonyls are all oriented in the same direction. Since the carbonyl carries a significant negative dipole there is a large electrostatic dipole moment induced along the alpha helix. Memorize how this arrangement relates to the N- and C-terminus of the helix. Is it the N- or the C-terminus of the helix that lies in the strongly positive potential region of the helix dipole ? Where would a negatively charged residue (such as a phosphate group) find a binding site: at the beginning or the end of a helix ? Helix Study a beta-sheet. Concentrate on how the alternating hydrogen bonds are formed between pairs of residuesin opposite direction. The example provided also has a cis-proline. Find it and study why the C-alpha atoms of the preceeding residue and of the proline are in cis, relative to the peptide bond ? Sheet Display only the backbone, and the side chain atoms of Glu, Asp (color red) and Lys, Arg (color blue) residues in a protein. Pick out the salt-bridges in your structure.   Download a DNA molecule. Study the phosphate backbone connections. Study and remember the arrangement of the 5' and 3' hydroxyl and the phosphate group. You should be able to discern the sequence of a DNA molecule from viewing the structure. You should also remember which way the helix turns: DNA is a right handed helix (except Z-DNA which is left-handed). B-DNA A-DNA Z-DNA Study the catalytic triad in a serine protease. In this structure (one of the classics: bovine trypsin in complex with the pancreatic trypsin inhibitor, by Robert Huber and Johann Deisenhofer, 1982) the triad is serine 195, histidine 57 and aspartate 102. 2PTC.pdb Study the ATP and t-RNA binding sites in a tRNA synthetase. Note the relative sizes of nucleotide structures and proteins. Pay special attention to the way the anticodon loop is tightly bound, as well as the acceptor stem. Using stereo, it should not be difficult to pick out the AMP molecule bound in this structure of E. coli glutaminyl tRNA synthetase right in the middle. 1EXD.pdb

Representation of Structures RasMol allows a number of different representations of the molecule, they are accessible through the Menu, but they can also be entered through the command line and you need to know the equivalences, in order to construct scripts. Note that these commands also accept Boolean values as arguments i.e backbone off is a legal command while backbone on defaults to backbone 0. Note that the numerical parameters are in RasMol internal units of 1/250 Å. Command line equivalences Display Menu Command Line Wireframe wireframe 0 Sticks wireframe 100 Backbone backbone 100 Spacefill cpk on or spacefill on Ball & Stick wireframe 50 CPK 120 Cartoons wireframe 100 for strands cartoon 380 for helices and strands Note that the command cartoon is not documented in the help feature ! Note that water molecules do not show up in a wireframe view. In order to see single, non-bonded molecules, you have to select them (see below) and then display them as CPK spheres. reset set stereo -5 set background [170,170,255] select all wireframe off backbone off center rotate z 133 rotate y 24 rotate x 42 backbone 80 color white select within (8.0, hoh1) OR within (8.0, hoh2) backbone off wireframe 80 select (within (3.5, hoh1) OR within (3.5, hoh2)) AND *.o?? color [255, 0, 0] select (within (3.5, hoh1) OR within (3.5, hoh2)) AND *.n?? color [0, 100, 255] select hoh1 label .HOH 1 select hoh2 label .HOH 2 select hoh1, hoh2 color [255,80,0] cpk 300 select all Script commands to show the two internal, structural water-molecules in 2IMM together with the residues in an eight Å radius around them. Hydrogen bonding atoms are colored.

Selection RasMol has a very powerful command-line syntax to select parts of the protein structure. This is the most useful part of the program - since it makes real work far more efficient than having to use the mouse, once you understand the syntax and commands - but it is also one of the most confusing features of the priogram. It is a good idea to have a reference sheet handy. Load the structure for 1A6M.PDB for the next example. You can do this either with RasMols drag-and-drop feature or with the File - Open menu. reset set background black set stereo -5 select all rotate z 174 rotate y -71 rotate x 107 translate x 13 translate y -3 zoom 129 wireframe off cartoon off color [180,180,180] backbone 100 select helix color [120,120,120] backbone off cartoon on select hem color [200,0,40] wireframe 100 select *.fe center color [100,75,0] cpk 320 select hem.o1 or hem.o2 color [255, 60, 0] cpk 200 wireframe 80 select (his64;B or his93) and (sidechain or his.ca) color cpk wireframe 100 A set of script commands to demonstrate selections with a view of myoglobin in the 1A6M structure. Here are the expression examples from the Quick Reference. Try some. Are you unsure sure how a specific atom is named? Just click it. Examples for expressions Command Meaning * All atoms cys Atoms in cysteines hoh Atoms in water molecules as? Atoms in asparagine or aspartic acid *120 Atoms at residue 120 of all chains *p Atoms in chain P *.n? Nitrogen atoms cys.sg Sulphur atoms in cysteine residues ser70.c? Carbon atoms in serine-70 hem*p.fe Iron atoms in the Heme of chain P *.*;A Atoms in alternate conformation A */4 All atoms in model 4 - this refers to multiple NMR-structure models in a single PDB file. Note that you can combine commands with Boolean expressions - and, or and not ! For instance: select (ser OR thr) AND NOT helix is a valid selection. Note that AND in the logical sense is not used how you would colloquially use it: if you want both serine and threonine, you need to specify ser or thr. Writing ser and thr means that both ser as well as thr have to be true at the same time. This would select nothing. Restricting is a concept very similar to selection, but it removes all parts that are not selected from the display. Try the following script. This also illustrates the useof the center command to center rotation to the center of gravity of a selection. restrict hem color [200,0,40] wireframe 100 zoom 500 center hem.fe select hem select *.fe center color [100,75,0] cpk 320 select hem.o1 or hem.o2 color [255, 60, 0] cpk 200 wireframe 80 Script to focus on the heme group of myoglobin.

Color Color can be entered either as a keyword, such as white or green, or as a triple of RGB values from 0 to 255 in square brackets e.g. [255,100,0] for orange. A number of predefined color schemes exist, that will color residues or atoms according to their properties. Important examples are cpk     Color atoms by chemical type. group    Color ramp from N-termuns (blue) to C-terminus (red). Note: if you cannot see the full color range in your protein, switch off display of heteroatoms and color again. temperature    Color by B-value (disorder or mobility) Other color choices are documented with the help function.

H-Bonds H-bonds are drawn between the donating nitrogens and the accepting oxygens. Alternatively, in a backbone-only view, they can be drawn between C-alphas with set hbonds backbone. The commands for hydrogen bonds and disulfidebonds (ssbonds true) are used in a similar way. reset backbone off cartoon off set background black restrict protein and backbone wireframe 120 color [180,180,180] hbonds 50 color hbonds [0,255,100] H-bonds of the Myoglobin backbone. Note that H-bonds are drawn for backbone atoms only - use monitor <atomnumber> <atomnumber> to draw lines between arbitrary atoms.

Measurements Use the commands set picking distance, set picking angle and set picking torsion for measurements. The first, set picking distance, will get the distance between the next two atoms you pick with your mouse. The second will get the angle between the next three atoms and the third will calculate the dihedral angle between the next four atoms you pick. Type set picking off to return to the normal mode again. Try this: here are three amino acids from myoglobin. Get the PHI and the PSI angle for the residue in the middle ! reset backbone off cartoon off wireframe off set background black rotate z -168 rotate y 49 rotate x 135 translate x 56 translate y -16 zoom 500 restrict 23-25 wireframe 70 color cpk center his24.ca zoom 300 set picking torsion Three aminoacids for picking and measuring backbone dihedral angles. Verify you measurement with the following command select his24, show phipsi and confirm this with show ramprint. See the help section of the show command, help show, to find what other information is available.

Surfaces and Slabs Rasmol can display van der Waals and solvent accessible surfaces and color them according to properties. Try the following for illustration: reset set background black set stereo -5 select all wireframe off cartoon off center hem.fe rotate z 123 rotate y -36 rotate x -169 translate x 41 translate y -19 zoom 220 restrict hem color cpk wireframe 90 cpk 150 dots 300 The Van der Waals surface of the heme prosthetic group of myoglobin. A "slab" or clipping plane allows you to see cross-sections of a molecule. Try this view: reset set background black set stereo -5 zoom 80 select all cartoon off backbone off wireframe off dots off cpk 400 color [170,170,240] select hydrophobic color [255,190,0] select hem color red slab 50 set slabmode solid A cross-section of Myoglobin shows the (red) heme-group packed into the hydrophobic interior (yellow). You can move the slab forward and backward by pressing [control] [command] and the mouse on the Mac, [control] left-mouse-button on the PC. type help set slabmode and try out the different modes to view sections.

Saving and Printing From the menu. Use write script <filename> to save a current view ! You can open, read and edit a script with any text-processor. Remember to save in text-only format when you are done. Images can either be exported in a number of formats, or just copied (Edit - Copy from the Menubar) and then pasted e.g. into a MSWord document. The File - Print command will print the current view.

Scripts We have been using scripts all throughout this tutorial. But RasMol can also export its current state to a file, as a script. RasMol generated scripts are invaluable to determine the rotation and translation paramaters of a specific view. Use write script <filename> to have all the transformations and commands required to generate your current view dumped into a textfile. If no path is specified, the file will be in the directory from which RasMol was started. You can open this file with a text-processor and then copy and use or modify the required transformations. Other than that, the file writes the view on an atom-by-atom basis and is therefore not really suitable to study command syntax or as a source for editable scripts.

Web Resources A collection of useful resources for RasMol on the Web. WWW Resources Site Description PDB The repository of protein structure coordinates. CATH Class, Architecture, Topology, Homology - a protein structure classification. SCOP Structural Classification of Proteins. HIC-Up Hetero Compound information database in Uppsala. Structures and data for the "other molecules", substrates, inhibitors, prosthetic groups etc. in the PDB. Tutorial A RasMol tutorial by Gale Rhodes, University of Southern Maine. RasMol The so called RasMol homepage at the University of Massachussets, by Eric Martz. Currently the site is pushing heavily for the author's CHIME implementation of a "protein explorer" which does not run natively under Unix operating systems. But there is still useful RasMol information too, especially a number of links to tutorials and scripts, if you search a little.

Feedback Has this been helpful ? Inaccurate ? Confusing ? Do you have suggestions for improvement ? Do any of the topics need to be expanded ? Did you come accross an interesting resource on the Web ? I welcome all feedback to boris.steipe@utoronto.ca.

INDEX BCH441 COURSE HTML TUTORIAL BIOINFORMATICS

B. Steipe and Program in Proteomics and Bioinformatics, University of Toronto boris.steipe@utoronto.ca Last revision: Sept. 2004 ©