Exploring Molecular Structures with RasMol

March 22, 2024 Off By admin

Table of Contents

Introduction to Molecular Visualization

Molecular visualization is the representation of molecular structures and dynamics using graphical models. It plays a crucial role in understanding the structure, function, and interactions of biological molecules such as proteins, nucleic acids, and small molecules. Visualizing these structures helps researchers and scientists in various fields, including bioinformatics, structural biology, and drug discovery, to interpret complex data and generate hypotheses.

Visualizing molecular structures provides insights into the 3D arrangement of atoms, which is essential for understanding how molecules interact with each other and with other molecules in their environment. This information is critical for studying protein folding, molecular docking, drug design, and many other biological processes.

RasMol is a popular molecular visualization program developed by Roger Sayle in the early 1990s. It was one of the first programs to provide interactive 3D visualization of molecular structures on personal computers. RasMol allows users to manipulate and analyze molecular structures, such as proteins and nucleic acids, in real-time. Its significance lies in its user-friendly interface, which made it accessible to a wide range of researchers and students. RasMol also played a key role in the development of other molecular visualization software and standards.

Overall, molecular visualization, including tools like RasMol, is essential for understanding the complex world of molecules and is a valuable tool in various scientific disciplines.

Using RasMol as a Computer Visualization Tool

Rasmol program is a simple, yet powerful tool, which enables you to visualize a molecule in “3-Dimensional space”. Since you actively manipulate the computer mouse to rotate the molecule in computer space, you develop a sense of the 3-dimensionality of the molecule. Through RasMol, you can also change the display format of the molecule to display different features of the molecule.

Through this tutorial, you can become familiar with this program to enable them to further explore the structure/function relationship that is a focal point in biochemistry.

At the end of this section, you should be familiar with how to use RasMol as a computer visualization tool. We will introduce you to the basic features of RasMol, including how to:

Organize your files
Start RasMol
Open a PDB File
Change the Background Color
Change Display Format of the Molecule
Use the Command Line to Change Formats
Change Colors
Identify Features within the Molecule
Select Features within the Molecule

Organize Your Files

- We recommend that you maintain separate folders on your computer (desktop or preferred disk drive location) for separate projects.
  - For instance, if you are using RasMol for SMART Teams, you should have a separate folder for each of these projects. Within these “larger folders”, keep a separate folder for themes, subjects or projects. This will enable you to keep subjects separated and will become important if you do any design work or generate script files, a way to save your work within RasMol (see RasMol Training Part II).
- In each folder, place a copy of RasMol and the PDB file with which you are working.
  - As you become more familiar with the program and begin to develop script files, you will probably have several folders with PDB files and script files. RasMol is a small program and it is okay to make multiple copies of it within your computer. We recommend that for each new project that has a new PDB file, you place a copy of RasMol in that folder. This will become especially important as you use RasMol to explore more molecular structures.
  - You can download a copy of RasMol from Rasmol.org

Opening RasMol

- To open the RasMol program, double click on the RasMol icon in your folder:

RasMol 2.6.5.1.exe

RasMol has Two Screens

- - You will notice that when you open the RasMol program, a window automatically opens with a black background on your computer monitor. In addition, there will be two buttons that appear on the Task Bar (see figure below). This is because there are two windows associated with RasMol: a Command Window and a Visualization Window.

Note: These directions are specific for PC-users. Macintosh users will utilize RasMac, will have the essential components described herein, but may also have differences.

Please see the appendix for Macintosh-specific directions.

When you launch RasMol, this is an image of what will appear on your monitor. Notice the two buttons on the task bar. One button will open the command window (the one on the left) and the other button will open the visualization window (the button on the right; this window will open automatically upon launching of RasMol). Open both of these windows and arrange them on the monitor so that you can see both of them on the screen.

Molecule Visualization Window

In order to work most effectively with RasMol, we recommend that you organize your screen to display BOTH of these windows. To open the Command Line Window, click on the RasMol Command Line button on the task bar. Arrange the Molecule Visualization window and the Command Line window so that you can see both windows and they are not overlapping. You can resize the windows to accommodate your preferences or computer monitor size. You will want the Computer Visualization window to be larger.

PDB Files

- RasMol is a molecular visualization program that enables us to visualize the atomic coordinates of a molecule that has been crystallized.
- These coordinates are stored in a file called a PDB (protein data bank) file.
- PDB files can be located and downloaded for free from the Protein Data Bank website (www.pdb.org).
- More information about saving a PDB file and understanding the information contained within the file will be discussed within RasMol Training Part II.
- For initial exploration of RasMol within Section I of the RasMol Training Guide, example PDB files that are used within this tutorial can also be found on our website at:
  - http://www.rpc.msoe.edu/cbm/smartteams/resources.php

Opening a pdb file

- After both windows of RasMol are opened and arranged on the screen, use the Pull-Down menu bar located on the Molecule Visualization window and chose “File.”
- Click on “Open” in order to open a PDB file
  - This will bring up a window entitled “Select Molecular Coordinate File” that will allow you to

direct the program to open a specific pdb file.

- - Click on the appropriate pdb file that you wish to open.
  - NOTE: Organizing your design work will be simplified if you always do the following: Create a RasMol folder with the RasMol program

and the PDB file, open the RasMol program by clicking on the RasMol icon inside the folder, use the pull down menu to open the PDB file with the commands >File>Open.

Default Display of Molecule

After you open the PDB file, the molecule will appear in wireframe format in the Molecule Visualization window. The wireframe format is the default setting and every time that you open a file, this is the initial format you will see. In the wireframe format,

the thin wire represents the bonds between each of the atoms. We will change the format in a moment.

Commands within RasMol

- There are two ways to enter commands and change the display of the molecule in the RasMol program:
  - One way is to use the Pull-down menu and select specific commands (like we just did with opening the PDB file).
  - The other way is to type specific commands into the Command Line window.
    - In the Command Line Window, you will see the prompt “RasMol>”. By typing commands at this prompt, RasMol is directed to do certain things. The commands that are entered at this prompt need to be specific. We will discuss this specificity in more detail as we progress through the training guide.
  - For the remainder of this section in the training guide, a combination of commands (both in the command window and the pull-down menu) will be used.

Closing a PDB file

- If you have completed work on one PDB file and would like to work on another file, you need to close the PDB file that you are presently working on before you can open a new PDB file. RasMol will not open another PDB file over the original. You must close the current file to open the next one.
- There are two ways to close a PDB file: using the Pull-down menu or using the Command Line window
  - Using the Pull-Down Menu
    - To close a PDB file, use the Pull-Down menu bar located on the Molecule Visualization window and chose “File.”
    - Click on “Close” to close the PDB file.
  - Using the Command Line window
    - To close a PDB file, type “zap” at the prompt.
      - RasMol>zap

Background Color of Molecule Visualization Window

The default setting for the Molecule Visualization window will be a black background. This can be modified and you can select any color for the background. Dark colors are not recommended if you wish to print an image of your molecule.

- To change the color of the background:
  - RasMol> background white
    - This will change the background of the window to white.
  - RasMol> background green
    - This will change the background to green.
      - This is obviously not a very nice color at which to look for most people, but for some reason, some people like this color.
  - RasMol> background blue
    - This will change the background to blue.
    - You get the idea. You can change the color of the screen to any color that you wish. We recommend white, as this has been the color that has worked the best for the most people. It also serves as the best backdrop if you export a picture (*.gif) so that you do not have a black (or green) background for your model. This saves on ink when printing, especially for posters. (See Number 14 in this section of the training guide for how to export an image of your molecule.)
    - Note: We recommend that you avoid the red/green color combination in case you, or others, have problems with color blindness.

Rotating the Molecule

- To rotate the molecule within the X-Y axes on the computer screen, place the mouse cursor in the Molecule Visualization window, hold down the left mouse button. Moving the mouse will rotate the molecule about the X-Y axes
- To relocate the molecule within the Molecule Visualization window, (for instance, to center the molecule within the window), place the mouse cursor in the Molecule Visualization window, hold down the right mouse button and move the molecule to the desired location.
- To zoom in or out, place the mouse cursor in the Molecule Visualization window, hold down the left mouse button AND hold the shift key. Moving the mouse will then zoom in or out on the molecule.
  - A word of caution: if you zoom in or out too fast, RasMol may close (crash) unexpectedly, as the program has to re-render the image at each step. Zooming too quickly, especially with large molecules, will overwhelm RasMol and the program will crash.
- To rotate the molecule within the Z axis, place the mouse cursor in the Molecule Visualization window, hold down the right mouse button and the shift key. Moving the mouse will then rotate the molecule about the Z axis.

Table Summarizing Mouse Button/Key Needed for Actions within RasMol

Action	Mouse Button (and Key) Needed
Rotate X-Y	Left Mouse Button
Move Molecule	Right Mouse Button
Zoom In/Out	Left Mouse Button & Shift Key
Rotate Z	Right Mouse Button & Shift Key

To Change the Format of the Molecule
- There are two ways to change the format of the molecule. One is through the pull-down menu and one is through the command line. We will do both so that

you are familiar with both aspects. As you become more comfortable with the program, we do recommend that you use the command line rather than the pull- down menu.

- - Using the command line allows you to add or subtract specific features one at a time. If you have created a design using the command line and then use the pull-down menu, the pull-down menu will overwrite all of the molecule features that you have created. This is more important as you develop more sophisticated models with specific features highlighted. In Section III of this RasMol Guide, we will do some exercises that will illustrate this point.

Changing format with the Pull-Down Menus

- Under the Pull-down menus in the Molecule Visualization Window, there is a “Display” option (see figure below). Within this pull-down menu, there are several different formats

that you can choose to display your model:

- - Wireframe
  - Backbone
  - Sticks
  - Spacefill
  - Ball and Stick
  - Ribbons
  - Strands
  - Cartoon
- Each of these display options will render your molecule in a different format. Some of these options will look new to you and some of them will look very familiar. When we start talking about designing a model for building purposes, we will talk more about which options are available for building, but for now, we are simply talking about using computer visualization programs and therefore we will cover each of the formats.
  - Each option has its advantages and disadvantages; which option you choose to display your molecule depends on which story you wish to tell. The advantage of using RasMol within the classroom is that it enables you to represent the model in a variety of ways, thus illustrating different aspects of the molecule.
- Let’s look at each of these in turn and see what the different formats represent.
Please note that the images to the right are of a single zinc finger and are based on amino acids #4-31 of the PDB 1ZAA.pdb.

Wireframe

- - - This is the RasMol default format for displaying molecules initially opened in RasMol. The thin wire represents the bonds between each of the atoms and the ends of the wires represent the atoms. The advantage of the wireframe is that all of the atoms are displayed. It is difficult to distinguish secondary structures in this format.

Backbone

- - - The alpha carbon backbone format only displays the position of the alpha carbon in each amino acid by a bend in the backbone. All of the other atoms within the amino acid are not displayed. The advantage of this display format is that it clearly illustrates the secondary structures within a molecule.

Sticks

- - - This display format is similar to the wireframe format, but rather than a thin wire, the atoms are connected by a stick. As in the wireframe format, all of the atoms in the molecule are displayed.

Spacefill

- - - In this format, each atom is displayed as a sphere representing the volume that atom occupies in space. For example, hydrogen atoms are smaller than carbon atoms, which are smaller than sulfur atoms. This format is advantageous in that the spacefill model represents the volume and three- dimensional shape of a molecule. Identifying the secondary structures or any internal atoms in this display format is challenging.

Ball and Stick

- - - In this format, each atom is represented by a small sphere (ball) and the bonds connecting each atom are indicated by a stick connecting the spheres. If the molecule is very large, this representation can become too overwhelming and specific information about the molecule can be difficult to determine.

Ribbons

- - - This format shows the molecule as if it were traced with a ribbon. This version may be familiar as it is often seen in textbooks. Secondary structures are easily identified within this format with the alpha helix represented as a coiled structure and the beta strand represented as a pleated structure.

Strands

- - - This format is similar to Ribbons, except that rather than being a solid ribbon, there are several “strands” or thin lines that make up the model.

Cartoon

- - - This format is similar to Ribbons, except that the beta strands are indicated by arrows to point out the directionality of the strands. This version is also widely seen in textbooks.

Format	Advantage	Disadvantage
Wireframe; Sticks; Ball and Stick	Displays all atoms	Difficult to distinguish secondary structures; too busy for large structures
Backbone	Secondary structures clearly seen	Detail of amino acids is lost, unless specifically added
Spacefill	Represents 3D volume and shape	Cannot identify secondary structure or internal atoms
Ribbons; Strands; Cartoon	Secondary structures are easily visualized; cartoon shows directionality of beta strands	No structural details

Changing display format with the Command Line

- In the command window, you can change the format of the model by typing the following commands:
  - RasMol>Backbone
  - RasMol>Wireframe
  - RasMol>Spacefill
- To generate the Ball and Stick image, you would use a combination of the wireframe and spacefill commands.
- In contrast with the Pull-down menu in the Molecule Visualization window, in the Command Line window, you can also alter the size. When we get to the model design stage of this training (in section III), this will become especially important. For now, we will just vary the sizes so that you can see what we mean by changing the size of the different formats. Following the format command, you enter a value for the size. Each format as a range of values, usually between 1 and 750. For scaling purposes, 1 RasMol unit equals 1/250 Angstroms.
- Enter the following commands in the Command Line window to see the effects of the commands:
  - RasMol>backbone 300
  - RasMol>backbone 30
  - RasMol>spacefill
  - RasMol>spacefill 100
  - RasMol>wireframe 200
  - RasMol>spacefill 300
- If you want to turn off a particular format, you would enter the following command
  - RasMol>spacefill off
  - RasMol>wireframe off

Changing color with Pull Down Menu

- RasMol has several color display options to make your model look visually pleasing, or to indicate specific features of the molecule.
Figures to the right are of hemagglutinin, a protein found on the flu virus membrane and are based on 2HMG.pdb.

The most frequently used color options are:

Monochrome

- - - This option will color the entire molecule white. This is often a good starting point if you would like the model to have a white backbone with other colors highlighting different features (to be discussed in number 16).

CPK

- - - This color scheme was developed by Corey and Pauling and later improved by Kultin in which carbon is gray, hydrogen is white, oxygen is red, nitrogen is blue. This is the standard color code and is utilized in many textbooks.

Chain

- - - If a PDB file has multiple polypeptides, as seen in the hemagglutinin protein on the right, in which there are 6 chains, or in the protein hemoglobin, in which there are 4 chains, this color option will color each chain a different color.

Structure

- - - This option will color the secondary structures different colors. Magenta is used to highlight helices, yellow to indicate beta sheets and blue to indicate turns (this feature is not shown in this figure).

Exporting an Image from RasMol

- If you would like to create a *.gif image from RasMol, you can do so using the pull-down menu option “Export”

- This menu option will then provide you with several different graphic formats (.gif, .bmp, etc) from which you could choose to save your image. After you select which format you would like to export your image, a save window will appear and you will be asked to name your file and direct the computer to which folder you would like to save the image.
- The exported image that is saved will look exactly like the one that is on your screen, which means that if you have a blue background, your image will also have a blue background. This is the reason why we recommend that you change the background to white if you plan to export any images.

Identification Feature of RasMol

- One of the features in RasMol is an identification feature that enables you to determine the identity of an atom by placing the mouse cursor over the molecule and pressing the left mouse button. After you click the mouse button, a line will appear in the command window giving identity information about the point on which you clicked.
- The format of the information displayed in the Command Line window will be similar to the following:
  - Atom: CA 140 Group: Asn 19 Chain: B
    - This line gives you several pieces of information:
      - The atom is the alpha carbon (CA) and it is assigned atom number 140.
      - This alpha carbon belongs in the Asparagine amino acid, which is the 19^th amino acid in this PDB file [Note: the amino acid number in a PDB file does not always correspond with the amino acid in the protein.]
      - The atom is in chain B of the molecule.
    - This information will be useful in several ways, which we will discuss later in our training.

“Select” command

- You can specifically select certain features within your molecule when using RasMol.
- There is a set of predefined terms that are recognized by RasMol (see Quick Reference Guide for full listing) http://www.rpc.msoe.edu/cbm/resources/pdf/refcard.pdf
- Once you select a portion of the molecule, all subsequent commands will apply to that selected portion. RasMol will not know that you meant to select another subset, or that you were done with that set of features, and so you must be very specific with your commands.
  - Examples:

Secondary Structures

RasMol>select helices
- This command will select all of the helices within the molecule.
After you have selected the helices, you can enter a command to change the format or color, and this command will ONLY be applied to the helices.

o For Example: RasMol>select helices

RasMol>color red

This series of commands will color the helices red.

- NOTE: At this point, you have selected the helices. If you enter any other commands, such as a new color, or display format, all commands will be applicable to JUST the helices. You will need to select a new subset of features, or “select all” to get out of the helix-selected mode.
RasMol>select sheets
- This command will select all of the beta sheets within the molecule.
- As we did with the helices, you can color the sheets any color, or change the format in which you have the beta sheets displayed.

Heterologous Groups

RasMol> select hetero
- This command will select all of the hetero atoms that are contained within this file. Remember that these are the atoms that are NOT amino acids. Examples include heme groups, zinc atoms, ADP and water.
- If you know the name of the group, you can select it directly by its name (such as “Select Zn”)

Amino Acid Sidechains

Within any molecule, you may wish to display all of the amino acids, or a specific set of amino acids (such as all of the hydrophobic amino acids), or only a select few amino acids based on function (such as the amino acids that make up an active site of an enzyme). Through entering commands in the Command Line window, you can make these selections.

RasMol Exercise

Let’s practice selecting amino acids:
- Open a PDB file in RasMol
  - Example PDB files can be found http://www.rpc.msoe.edu/cbm/smartteams/resources.php
- Change the background color to white
- Change the display format to backbone
- To select all of the amino acids:
  - RasMol>select sidechain
  - RasMol>spacefill 275
  - RasMol>wireframe 225
- To select a subset of amino acids, you can use predefined terms, such as “polar” or “hydrophobic” to display all of the amino acids that are in these categories.
Note: There is a list of predefined terms that RasMol recognizes on the RasMol Quick Reference Guide.
- - To select all of the polar amino acids:
    - RasMol>select polar
    - RasMol>color red
  - To select all of the hydrophobic amino acids:
    - RasMol>select hydrophobic
    - RasMol>color yellow
  - You can also select all of particular amino acid, such as all of the histidines in a molecule.
Note: RasMol recognizes the three letter abbreviations for the amino acids.
- - - RasMol>select his
    - RasMol>color magenta
      - This allows you to identify all of the histidines within the molecule. By moving the mouse cursor over these magenta regions, you can click on them to identify which histidine the amino acid is in order to determine the amino acid number so that you can select this amino acid specifically.
  - To select functionally significant amino acids, you could refer to the primary citation associated with the PDB file (more on that in Part Two of the training). If you know that you want to display the sidechains for a particular amino acid, for example

Histidine 63 in the beta-globin protein, you would select that amino acid specifically.

- - - RasMol> select His63
      - Note: Although we typically write His 63 with a space between the letters and the number, there is not a space between the letters and number when writing a command in RasMol.
    - RasMol>spacefill 275
    - RasMol>wireframe 225
      - These commands will generate a ball and stick representation of all of the atoms in the amino acid. Please note that ALL of the atoms within the amino acid are displayed as balls and not just as the sidechain atoms. This is a “bumpy backbone” versus a “clean backbone” and we will discuss this difference in Section III of the RasMol Training Guide.
    - RasMol>color CPK
      - This command will color the sidechains CPK.

Changing color with Command Line

- To change the color of the model with the command line, there are two options: to use the predefined color list (refer to Quick Reference Guide) or to use the RGB color numbers (again, use the Quick Reference Guide, or any source that indicates RGB [computer color system of Red, Green, Blue] color numbers).
  - RasMol> select helices
  - RasMol> color red OR RasMol> color [240,0,0]

Conclusions for Section I

Hopefully at the end of this section, you feel comfortable:

- starting RasMol on your computer
- opening a PDB file
- changing the background color
- changing the display format of the molecule
- changing the colors of the molecule
- selecting certain features of the molecule
- using the pull-down menu
- using the command line

Through these features, we encourage you to use RasMol within the classroom as a visualization tool to enhance your lessons on biochemistry.

Additional Exercise

Exercise I

Start RasMol and open Zinc Finger.pdb in RasMol. (can download this file at http://www.rpc.msoe.edu/cbm/smartteams/resources.php)
Change the background color to white.
Display zinc finger in the alpha carbon backbone format.
Display Zinc ion in relation to the zinc finger
Display sidechains for the histidine and cysteines coordinating the zinc ion
Display the sidechain for Phe16
Display the sidechain for Leu22
Display the sidechain for Arg18

Exercise I Answers

Start RasMol and open Zinc Finger.pdb in RasMol.
Change the background color to white. RasMol>background white
Display zinc finger in the alpha carbon backbone format. RasMol>backbone 300

RasMol>wireframe off

Display Zinc ion in relation to the zinc finger RasMol>select zn

RasMol>spacefill

Display sidechains for the histidine and cysteines coordinating the zinc ion RasMol>select his

RasMol>spacefill 275

RasMol>wireframe 225 RasMol>select cys RasMol>spacefill 275

RasMol>wireframe 225

Display the sidechain for Phe16 RasMol>select phe16 RasMol>spacefill 275 RasMol>wireframe 225
Display the sidechain for Leu22 RasMol>select leu22 RasMol>spacefill 275 RasMol>wireframe 225
Display the sidechain for Arg18 RasMol>select arg18 RasMol>spacefill 275 RasMol>wireframe 225

RasMol Training Section II: Understanding the Protein Data Bank and More Specific Commands within RasMol

In Section II of the MSOE Center for BioMolecular Modeling RasMol Training Guide, the focus is to learn about the Protein Data Bank, the worldwide repository for the crystal structure files, and to learn additional specific commands within RasMol. Section II will emphasize how to use RasMol in a more sophisticated manner using these specific commands. Mastering the material in this section will prepare you for further understanding of how to use RasMol and using RasMol in the Protein Modeling Challenge in the MSOE Center for BioMolecular Modeling sponsored event in the Science Olympiad (www.rpc.msoe.edu/cbm/scienceolympiad). In this section, we will focus on how to

- Use the Protein Data Bank (www.pdb.org)
- Save your work on RasMol by generating script files
- Open script files in RasMol
- Edit script files
- Center molecules in RasMol
- Select whole fields within RasMol
- Restrict subsets of the molecule within RasMol
- Add and remove hydrogen bonds within the molecule in RasMol
- Add and remove disulfide bonds within the molecule in RasMol

Part I

Protein Data Bank

- The PDB (www.pdb.org) is the single worldwide repository for the processing and distribution of 3-D structure data of large molecules of proteins and nucleic acids.
- As of 25 September 2008, there are 53,263 structures within the PDB.
- Resources within the PDB:
  - Molecule of the Month http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_th e_month/index.html
  - PDB Newsletter by Gary Graper describing the Science Olympiad http://www.rcsb.org/pdb/general_information/news_publications/newslett ers/2005q2/education_corner.html
  - PDB Newsletter (read about the New Jersey Science Olympiad Protein Modeling Challenge Event sponsored by the PDB) http://www.pdb.org/pdb/static.do?p=general_information/news_publicatio ns/news/news_2006.html#20060404
  - Educational Resources

http://www.pdb.org/pdbstatic/education_discussion/educational_resources

/education_flyer.pdf

- Each entry within the PDB contains several pieces of information:
  - Structure summary page
    - PDB Structure ID number
      - This 4 letter/number ID is a unique identifier that is assigned to the crystal data file upon deposition into the database.
    - Title
      - Title of the PDB file

- - - Authors
      - These are the researchers who were involved with the crystallization of the molecule. Note: The senior author or principal investigator is usually the last author in science publications.
    - Primary Citation
      - The journal article that accompanies the PDB file; excellent research resource for understanding the function of the molecule.
    - History of deposition and release
      - The date that the molecule was deposited into the PDB and the date the information was released to the public.
    - Method of structure determination
      - The method that was used to obtain the structural data (ex: NMR, X-ray diffraction).
    - Resolution at which the molecule the structural data was collected
      - How accurate the data is; the smaller the number, the better the data.
    - Molecular Description
      - This will tell you the number of chains within the molecule and the chain identity; for example in the hemoglobin file (1A3N.pdb), the chains A and C are the alpha-globin molecules and chains B and D are the beta- globin molecules.
    - Functional Class of the molecule
      - What type of molecule is it? (Ex: a toxin, an enzyme)
    - Source of the molecule
      - From which species was the molecule isolated? (human, bacterium, virus, mouse)
    - Chemical component
      - In this section, you will be able to determine if there are any heterologous groups that were crystallized with the molecule. Not all PDB files will have this section.
      - The 2-3 letter identifier used to designate the chemical components contained within the file listed are recognized by RasMol.

o For example, if this section stated that there was NAG (N-acetyl-glucosamine) contained within the molecule, RasMol would recognize “NAG” and you could therefore “select NAG” and RasMol would be able to select the atoms within that chemical component of the PDB file.

- - Sequence Details
    - This section of the PDB file provides specific sequence information as well as secondary structure information about the molecule.
    - In this section of the PDB file, you can identify where within the protein the alpha helix or beta sheets are located, as well as the amino/carboxy termini, which are the first and last amino acids of the protein, respectively (for more information about identifying these amino acids, please refer to number 13 within this section of the training guide).

Search the Protein Data Bank

3. Download a PDB file

To save a pdb file, click on “Download Files,” which is located on the left navigation bar

- One of the features of the PDB is the ability to search the database for files. You can search using key words or authors by entering these terms in the search box, highlighted in red in the figure below. Or, if you already know the PDB structure ID number, you can enter that number in the search field. After you have entered the search terms in the field, hit enter or click on the “search” button to the right of the search field.

Search Field

Practice searching:

In the search field, enter “aquaporin” and click on “search.”
- This search will bring back a search result that gives 18 structure hits.
- The PDB will list all 18 of these structures.
If you enter “hemoglobin” into the search field, you will see that there are 362 structure hits.
- If you do a broad inquiry like this one, you will need to look through the structures to see which one meets your requirements, for example:
  - Human versus mouse
  - Mutant versus “normal”

Download a PDB File

To save a PDB file, click on “Download Files”, which is located on the left navigation bar.

This will create a pull-down menu that will give you several options:

Download Files PDB File PDB gz

PDB File (Header) mmCIF File mmCIF gz

mmCIF File (Header) PDBML/XML File PDBML/XML gz

PDBML/XML File (Header) Structure Factors File Structure Factors gz Biological Unit Coordinates



FASTA Sequence

Select this option. By clicking on this link, it will bring up a “File Download” window. At this point, you click on the “Save” option and select which folder you would like to save the PDB file on your computer.

Remember our recommendation of organizing your work to include the PDB file and a copy of RasMol in a separate folder for each project on which you are working.

This organization will become important as you save your work (generate script files), which will be discussed in number 8 in this section of the training guide.

Searching the PDB and Reading a Structure Summary Page (practice)

- Search the database for “SARS spike receptor binding domain coronavirus protein”
- 8 structures should appear (as of 25 September 2008)
- Look for the one authored by Li et al and click on the PDB structure ID number
- You should arrive at the structure summary page for 2AJF.
- Use this structure summary page to answer the following questions about the PDB file (answers are at the end of this section):
  - Who are the authors of the PDB file?
  - In which journal was the primary citation published?
  - On what date was the file deposited into the PDB?
  - How many chains are in this file?
  - Are there any heterologous groups within this PDB file? If so, which ones?
  - From what source was this molecule isolated?

Download a File (practice)

- We are now going to work with the potassium channel as our model PDB file.
  - Create a folder labeled “RasMol Training”. Within this folder, create a Kchannel folder.
  - Place a copy of RasMol in this folder.
  - Search the PDB for 1J95
  - Download 1J95 to the K Channel folder on your computer
    - This organization will be especially important within this section of the guide.

Opening a PDB file (review)

(for more information see Section I)

- Opening PDB files
  - Double-click on the RasMol icon within your folder.
  - Arrange both windows on your screen.
  - Go to the file menu.
  - Click on “Open.”
  - Click on the appropriate PDB file (open 1J95).

A little review from the previous section

- How do we do the following items?
(answers at the end of this section)
- - Change Background to white
  - Change display formats
  - Change colors
  - Highlight the helices as red

Save your work – Creating a Script File

- RasMol has a feature that allows for you to save your work. In order to save you work, you will generate a script file.

RasMol> save script filename.spt

Example: RasMol> save script Kchannel.spt
- We recommend saving multiple versions of your model design as you work.
  - For example, as you progress through your design, save Kchannel1.spt, Kchannel2.spt, Kchannel3.spt, etc. This allows for you to return to previous files, in case you accidentally save a file that you did not like, or if you make a mistake. There is not an “undo” command in RasMol.
  - Through saving multiple versions of your work, you can return to a previous version if you accidentally do something to your molecule that you do not like, or if you accidentally lose your work.
  - As with any important document, save your work often!
Please Note that it is very important to include the “script” and the “.spt” components to this command.
- If you enter the command with “save filename.spt”, you will create a PDB file and NOT a script file.
- If the save command does not include these items, a script file will NOT be created. If you exit RasMol before a proper script file has been created, all work that you have done will be lost.
  - Once you have entered this command, you have saved your work.
  - Check your folder for your newly created script file.
If you have organize your work in such a way that there is a copy of RasMol and the PDB file in the same folder, and have launched RasMol from this folder, the RasMol program will automatically save the script file to this folder.

Opening a script file

- To open a script file:
  - First, launch the copy of RasMol in your Kchannel folder.
  - In the command line window, enter the command:
RasMol>script filename.spt
- Example:

o RasMol>script kchannel12.spt

- - This should open your version of the molecule in the molecular visualization window.

Practice saving/opening a script file

- Open the Kchannel PDB file 1J95 using the pull-down menu.
  - RasMol>select all
  - RasMol>backbone 300
  - RasMol>wireframe off
  - RasMol>color white
  - RasMol>select helices
  - RasMol>color cyan
  - RasMol>select hydrophobic
  - RasMol>color yellow
  - RasMol>save script kchannel.spt
- At this point, we have created a script file, or saved your work. We have generated an image in which the helices are colored cyan, except where there are hydrophobic amino acids.
  - Question: Why are there so many hydrophobic amino acids located on the outside of this protein when we have learned that hydrophobic amino acids are typically located buried within the molecule, away from the aqueous external environment? (answer at the end of this section)
- Okay, now that we have generated a script file, let’s erase the image from the screen so that we can open the script file
  - RasMol>zap
  - RasMol>script kchannel.spt
- Did your original file open?
  - If so, congratulations, you have created your script file and opened your script file successfully! If not, go back and see where you made your mistake before continuing.
Did you have the “save”? Did you have the “script”? Did you have the “.spt”?
If the script file did not open, either the command was not entered correctly in the command line window, or the file was saved in a different folder location.

Editing your script file

- Any time that you generate a script file, RasMol will place the script file in the folder that RasMol was originally launched. This is the reason that we recommend organizing your work so that each molecule you are exploring has its own folder with a copy of RasMol and the PDB. Each time you generate a script file, it will then be saved in that folder.
- When you save your script file, a specific pathname will be generated and saved within the script file directing RasMol to look within a specific folder for that particular PDB file.
- There may be times in which you work on one computer and wish to transfer your file to another computer. If you simply copy or move the script file to a new

computer or a new folder and try to open the script file using the command described above, RasMol will be unable to locate the PDB file because it will be looking for the PDB through the specific pathname that was generated when the script file was generated.

- This can be circumvented by editing the script file.
- To edit your script file, open your script file using a word processing program such as Notepad or Wordpad. We recommend that you DO NOT use MS Word for this process.

This is what you will see when you open your script file:

#!rasmol -script

# File: kchannel.spt

# Creator: RasMol Version 2.6

zap

load pdb “D:\CBM\Summer 2006\RasMol Training 2006\RasMol Training II\1J95.pdb” background [255,255,255]

set ambient 40 set specular off

- The text in blue is the pathname and tells RasMol the folder location of the PDB file. If you wish to use this script file on other computers, you will want to edit this line in the script file so that you can open the script file on any computer. In order to open this file on any computer, simply change the pathname to what you see in the text box below in red:

#!rasmol -script

# File: kchannel.spt

# Creator: RasMol Version 2.6

zap

load pdb “.\1J95.pdb” background [255,255,255]

set ambient 40 set specular off

- By changing the pathname to “.\1J95.pdb”, you are making a generic pathname (ie: load the PDB file located in “this” folder). If you have a folder on the new computer with RasMol, 1J95.pdb and the script file, you will be able to open the script file. This generic pathname will work, only if the folder contains RasMol, the PDB file and the script file. You must launch RasMol from this folder for the generic pathname to work.
- After you have saved your script file and you know that you are going to be changing computers, we recommend changing the pathname. This is especially

important if you plan to email the sript file amongst team members. Once you change the pathname, you will not need to do it again.

RasMol Commands

- Section I of the MSOE CBM RasMol Training Guide introduced you to the basic commands within the RasMol program. We will now present additional commands to further develop RasMol design skills.
The “*” Command
- The * key within RasMol is wild card for a whole field in which you can designate all atoms within a field.
  - For example, if you wanted to select all of the atoms within Chain D of 1J95.pdb, you would type:
    - RasMol>select *d
      - This command would then select all of the atoms within chain D of the K channel.
Restrict
- This command allows you to restrict your view of the molecule to specific to portions of the molecule dictated by you. You can restrict to specific chains, specific amino acid sections or specific features.
  - For example, if you wish to only look at chain D in the K channel, you can type:
    - RasMol>restrict *d
      - Notice that we have used the “*” command to select all of the atoms in Chain D and we have used the “restrict” command to restrict our viewing of only the atoms in Chain D.
      - Note that with the restrict command, all of the other parts of the molecule have disappeared from view. The other atoms are still in the PDB file, but you have “restricted” your view to the specified region.
Center
- The “center” command will allow us to center the molecule around a certain portion of the molecule.
RasMol will by default center the molecule at the center of the entire molecule. If you restrict your viewing to a certain subset (as we did with the K channel to just Chain D), when we rotate the molecule around in space, the molecule will seem lopsided. This is because although we have restricted our viewing of the molecule to just Chain D, all of the atoms are still present, we just do not see them.
Therefore, we can use the “center” command to center the molecule on the restricted region.
You will need to enter this command every time that you open the script file.
- To center the K channel on Chain D, you can type:
  - RasMol>Center *d
    - Notice that once again, we are using the “*” command to dictate to RasMol that we are centering the molecule around atoms in Chain D.
- At this point, we will open another PDB file so that we can illustrate the remaining items that we will discuss in this section. If you wish to save your script file at this point, you may do so. Remember to zap the existing molecule so that you can open a new PDB file. Or use the file pull-down menu and select “close”.
- Please search the Protein Data Bank for 1HTM.pdb and download this file to your folder.
- Open 1HTM.pdb on your computer.
Change the background to white.
Change the display to backbone.
Hydrogen Bonds
- Hydrogen bonds are essential to the stability of secondary structures.
- To add hydrogen bonds to secondary structures within your molecule:

- - RasMol>hbonds
    - After you have enter this command, you will notice that there are dotted lines that have appeared. These are the hydrogen bonds.
    - Since these bonds are very difficult to see, let’s give them an added dimension.
  - RasMol>hbonds 225
    - Notice that the Hydrogen bonds are now thicker, but that they appear to be floating in air. This appearance results from the fact that hydrogen bonds

form between the atoms that make up the backbone of the amino acid (the nitrogen and the oxygen atoms), but since we have displayed only the alpha carbon atoms, it appears as if the hydrogen bonds are floating in space. Therefore, we must set the hydrogen bonds to the backbone.

Notice that the bonds are blue/red, as they

are based on the CPK color scheme since they connect an oxygen (red) to a nitrogen (blue).

- - RasMol>set hbonds backbone
    - Notice that now the hydrogen bonds are attached to the alpha carbon backbone. The bonds are now colored gray since they are connecting the alpha carbon backbone, which is gray (carbon), to another part of the alpha carbon backbone. When bonds connect different sections, they adopt the coloring of the section that they are connecting.
  - To turn off the hydrogen bonds:
    - RasMol> hbonds off
      - This command will turn off expression of all of the hydrogen bonds within the molecule, bringing you back to the original state of the molecule in which no hydrogen bonds were displayed.
Disulfide Bonds
Some molecules will have disulfide bonds present within the structure. These bonds are between two nearby cysteine amino acids. To visualize these bonds, we can type:
- RasMol>ssbonds
  - As we saw with the hydrogen bonds, simply typing “ssbonds” will only produce dotted lines. To give these bonds dimension, we must add a value (thickness) to the ssbonds.

- RasMol>ssbonds 225

- - Notice that this command gives the disulfide bonds a thicker dimension, but as we saw with the hydrogen bond, the bond is “floating” in space. This is because the disulfide bond is actually between the sulfur groups of the cysteine sidechains, and not the alpha carbons. To make the disulfide bond connect between the backbone units, we need to set

the bonds to the backbone. Note that the disulfide bond

is orange (the CPK color for sulfur).

- RasMol>set ssbond backbone

- - Notice that this command sets the disulfide bond to the backbone. Once it does this, the disulfide bond is now gray. RasMol will assign the bond to adopt the color of the backbone that the bonds connect. Since the backbone is gray, now the disulfide bond is gray. Since sulfur is orange in the CPK color scheme, we will enter a command into the command line

window to color the bond orange.

- RasMol>color ssbond orange
  - Note that we specified that we wanted to color the ssbond orange. We need to be specific that we want to color the ssbond orange. What happens if you type in “color orange”?
- You may wish to display the disulfide bond connected to the cysteine sidechains, rather than to the backbone of the molecule.
  - In order to set the ssbond between the cysteine residues, you need to selectively display the cysteines.
  - After you have these amino acids displayed, you can the set the disulfide bond to the sidechains, rather than to the backbone.
    - RasMol>set ssbond sidechain
    - This will place the bond in between the sulfur atoms of the cysteines, rather than the alpha carbon backbone, as we saw earlier.
- To remove ssbonds:
  - RasMol>ssbonds off

Identifying the Amino and Carboxy Termini

- An important concept in protein structure is that each protein has an amino terminus and a carboxy terminus. Through RasMol, each student can readily identify each of these termini.
  - Amino Terminus
    - The Amino Terminus is the first amino acid in the protein. When a protein is synthesized, it begins with the 5’ end of the mRNA and synthesizes in a 5’ to 3’ fashion. Therefore, the first amino acid in the protein will be the amino acid that is encoded at the 5’ end of the mRNA.
    - To determine the amino terminus of the protein in the PDB file, click on the atom at the end of the protein. The atom with lowest amino acid number will be the amino terminus.
    - Alternatively, you may search the PDB sequence information to identify the amino terminus amino acid and use the RasMol command line window to select the specific amino acid. (See Number 1, Sequence Details of the Protein Data Bank File).
  - Carboxy Terminus
    - The Carboxy Terminus, on the other hand, will be the last amino acid in the protein.
    - To determine the identity of the carboxy terminus, click on the atom at the end of the protein. The amino acid with the largest number will be the last amino acid in the protein
      - For example, in the 1HTM.pdb protein, in chain, the amino terminus is Ser 40 and the Carboxy terminus is Arg 153.

Conclusions for Section II

At the end of Section II, you should be comfortable with

- Searching the Protein Data Bank
- Reading a structure summary page
- Downloading a PDB File
- Saving script files
- Opening script files
- Editing script files
- The “wild card” (*) command
- Restricting a section of the molecule
- Centering the molecule
- Adding hydrogen bonds
- Removing hydrogen bonds
- Adding disulfide bonds
- Removing disulfide bonds
- Identifying the amino and carboy termini

Answers to Questions Posed within Section II

Searching the PDB and Reading a Structure Summary Page (practice)

- Use this structure summary page to answer the following questions about the PDB file (answers are at the end of this section):
  - Who are the authors of the PDB file?
    - Li F, Li W, Farzan M, Harrison SC
  - In which journal was the primary citation published?
    - Science
  - On what date was the file deposited into the PDB?
    - 1 August 2005
  - How many chains are in this file?
    - 4 (A, B, E, F)
  - Are there any heterologous groups within this PDB file? If so, which ones?
    - Yes
      - Zinc, Chloride, Mannose, N-acety-D-glucosamine
  - From what source was this molecule isolated?
    - Human

A little review from the previous section

- How do we do the following items?
  - (answers at the end of this section)
Change Background to white
- RasMol>background white
Change display formats
- Use the pull-down menu (display)
- Use command line window
  - RasMol>backbone
  - RasMol>wireframe
  - RasMol>spacefill
Change colors
- Use the pull-down menu (color)
- Use the command line window
  - RasMol> color red
Highlight the helices as red
- RasMol>select helices
- RasMol>color red

Question: Why are there so many hydrophobic amino acids located on the outside of this protein when we learn that hydrophobic amino acids are typically located buried within the molecule, away from the aqueous external environment?

Answer: The K channel is a membrane embedded protein and therefore will be in contact with a hydrophobic region of the membrane. As such, it is necessary for the part of the K channel that interacts with the hydrophobic portion of the membrane needs to be hydrophobic as well.

RasMol Training Section III:

Designing a Model to be Built on the Rapid Prototyping Machines

Through this section of the RasMol Training Guide, you will become familiar with the commands needed to design a model that will be built on the rapid prototyping machine. As you become more comfortable using RasMol, this section will enable you to take the next step and be able to become a model designer.

In this section, you will learn how to

- Select an appropriate display format to use in your model design
- Add monitor lines for structural support
- Use Boolean operators
- Add hydrogen bonds within the beta sheet
- Remove “triangle bonds” within beta sheets
- Add sidechains to create a “clean backbone”
- Determine the appropriate sizes for Z corporation printed models
- Select appropriate colors to be used for the Z corporation printer

Model Design

How do you choose which type of display format to use in your model design?

- This is a common question that is asked by model designers. And the answer to this question is another question: “What is the story that you are telling with your model?”
- If your story is focused on a particular active site within the molecule, then perhaps the alpha carbon backbone model displaying key active site amino acid sidechains is the best display format to choose. If your story is focused on how two subunits interface at the surface, then perhaps the spacefill format is the best choice. Ultimately the choice is yours. In Section I, there is a table highlighting the advantages and disadvantages of each display format. This may assist you in deciding which format is the best for telling your story.
- The important point to remember is that no one model will tell every aspect of the story. Using RasMol in combination with the physical model will assist you in telling multiple aspects of your story.
- The CBM will build models in the spacefill format, wireframe and the alpha carbon backbone format. Models in the alpha carbon backbone format can have sidechains and heterologous groups displayed. We currently do not have the ability to build models in cartoon, ribbons or strands format.

Monitor Lines: What are they and where to place them

- When a model is built on the rapid prototyping machine, it is done through a layering system. A layer of powder 1/1000^th of an inch thick is spread out. This powder is impregnated with droplets of binder with ink (Z corporation printer) or scintered together with a laser (SLS machine). At the end of the production time,

the loose powder not incorporated into the physical model is vacuumed away. During the build time, there needs to be support within the model in order to withstand the additional pressure that accumulates as the powder builds up.

Hence, monitor lines are added within the model for support.

- How do you know where to place Monitor Lines?
  - Monitor lines are NOT needed:
    - Within Beta Sheets
      - If a molecule has beta sheets within the structure, the presence of the hydrogen bonds within these sheets will provide very good support. Therefore, you do not need to add monitor lines within beta sheets.
    - Within Alpha Helices
      - Alpha helices are very stable internally, therefore, neither hydrogen bonds nor monitor lines are needed within the helix structure itself.
  - Monitor lines ARE needed:
    - Monitor lines will be needed in regions of the protein that look like they might be able to squeeze together.
      - A good example of this: If you look at the image below on the left, you can see the alpha helices are colored in blue. These helices are stable from top to bottom, as mentioned above, but they do have the tendency to flex from side to side. (See regions indicated by yellow brackets in the figure below, which is based on 1TIM.pdb.) Due to this flexibility from side to side, monitor lines are needed to stabilize the helices.
      - By adding monitor lines in between the helices, as shown in the figure below as yellow bars (and pointed to by the arrows), you will increase the stability of this protein and prevent the flexing of the helices.

Before monitor line addition

After monitor line addition

- - - - To determine if the model will need monitor lines, we do the “squeeze test.”

The “squeeze test” refers to the ability to potentially squeeze a portion of the molecule together. When you look at the image of the molecule on the computer screen, can you see potential regions where you might be able to squeeze the model together, or potential flex points? If so, then you need to add monitor lines to this region to prevent this “squeezing” from occurring.

- - - - We recommend that you add monitor lines at the “top” and “bottom” of the helix in order to anchor the helix. If the helix is exceptionally long, then adding a monitor line in the middle will further stabilize the helix.
    - Monitor lines are also needed where there are large loops and turns within a molecule that could potentially be sites that need additional support.

In the image on the left, the blue arrows indicate a loop that would need a monitor line to stabilize the loop. On the image on the left, the blue arrows indicate the positions in which yellow monitor lines have been added to stabilize the loops.

Before monitor line addition

After monitor line addition

- - - Monitor lines should also be used to connect heterologous groups, such as the zinc ion in a zinc finger, as shown in the figure below. (This figure is based on 1ZAA.pdb, which was used in Section I of the training guide.)
      - If heterologous groups are not connected with monitor lines, they will be built as a separate piece. For example, in the figure to the left there are not currently monitor lines attached to the zinc ion, so the protein component (gray) would be built as one unit and the zinc ion (green) would be a separate piece.

Zinc ion that is not attached with monitor lines.

Zinc ion that is attached with monitor lines.

- - - Monitor lines are also needed to connect subunits if your molecule has multiple subunits. If you do not use monitor lines to connect the subunits, the molecule will build as separate subunits that are not connected.
      - Below are two figures of fibrinogen, based on 1JY2.pdb, which has three chains, each a different color. The figure on the left does not have any monitor lines and if it were built in this fashion, it would be built as three separate chains. In the figure on the right, there are monitor lines (show in yellow and indicated by the arrows) that will hold the three chains together.

After monitor line addition

Final Note on monitor line placement:

Before monitor line addition

Monitor lines are used to stabilize the molecule. Anywhere that you think that the molecule looks like it needs additional support, add a monitor line.

Adding monitor lines

- Monitor lines are added to the molecule by entering in a command in the Command Line window.
- To add the monitor line, you must first identify the atom numbers for the atoms between which you intend to draw the monitor line. To do this, move your mouse cursor over the atoms and click the left mouse button. This action will generate information in your command line window that will provide you with the atom identity, as described in Section I. The first number that is provided in that line of

information is the atom number, and this is the number that you need in order to create a monitor line.

- - Once you have identified the two atom numbers between which you would like to have the monitor line exist, the next step is to enter the command to make a monitor line:
    - RasMol>monitor atom number atom number
      - Example: RasMol> monitor 656 8567

It is essential to put a space between the numbers.

It is also essential to use the atom number and not the amino acid number.

- - - - You will notice that monitor lines initially appear as a dotted line, just as the hydrogen bonds and disulfide bonds did (see Section II).
      - In RP-RasMol, to add dimension to monitor lines:

RasMol>set monitor 225

Please note that this feature is only available in RP-RasMol. You can create monitor lines in RasMol, but you can only add dimension to the monitor lines with RP- RasMol. To obtain a copy of RP-RasMol for academic purposes, please contact Tim Herman at [email protected].

- - - Once you have given the first monitor line dimension, you will not need to do this again. All future monitor lines will have the same dimension. All monitor lines will have the same dimension.
    - The monitor line command is similar to a toggle switch. If you decide that you do not like the position of the monitor line or if the monitor line is too long, you can simply re-enter the monitor line command (in the above example, RasMol>monitor 656 8567) and it will turn off the monitor line.
    - If you wish to turn ALL of the monitor lines off:
      - RasMol>monitor off

Please note that this command will turn off all of the monitor lines within the molecule. To turn off specific monitor lines, use the monitor command above.

- - - A number will appear next to the monitor line and this number is the length of the monitor line in angstroms. A monitor line should not be longer than 9 angstroms. If the monitor line is longer than 9 angstroms, it is not stable and it defeats the purpose of being present to stabilize the molecule.

Coloring the Monitor lines

- When the monitor line initially appears, it will be the color of the atoms that the monitor line is connecting.
  - If the two atoms are the same color, then the monitor line will be the same color throughout the length of the monitor line.
  - If the two atoms are different colors, then the monitor line will be half one color and half the other color.
- To specifically color the monitor line (as opposed to the default setting described above):
  - RasMol>select all
  - RasMol>color monitor white
- It is essential that you include “monitor” within your command. If you forget to include the “monitor” within the command, you will color your entire molecule whatever color you have selected for your monitor color (in the above example, white).
- We recommend that you choose a light color for your monitor lines, such as white or light gray. Monitor lines are support structures and should not be the emphasis of your model. A bright or dark color will draw the user’s eye to that feature and that should not be the focus of your model.
- We at the CBM often color monitor lines and hydrogen bonds the same color (white). Some SMART Teams in the past have opted to color their monitor lines a different color from their hydrogen bonds to differentiate the two types of structural features. This is entirely up to the designers. Our only recommendation is to downplay the color (light colors) choice in order to prevent the focus from being on these structural features.

Boolean Operators

- You can link together RasMol commands by using Boolean Operator (And, Or, Not) in order to select very specific things in RasMol.
- Boolean Operators

- - OR (RasMol>select A or B)
Selects everything in both circles
- - AND (RasMol>select A and B)
Selects only that which is in the region that overlaps between the two circles
- - NOT (RasMol>select A not B)
Selects the region in A that does not overlap with B
- Practice:
  - Round #1
Select men and women
Stand up
- NO ONE should have stood up (unless you are a hermaphrodite)
  - Round #2
Select men or women
Stand up
- EVERYONE should have stood up because this selection process selects everyone who fits the category Men as well as everyone who fits in the category Women
  - Round #3
Select women and Wisconsin residents
Stand up
- Only women who live in WI should have stood up
  - Round #4
Select men or those who are wearing black shirts
Stand up
- All men should have stood up, as well as women wearing black shirts
- Note that some may have fulfilled both aspects in that men with black shirts may be present.
- The use of Boolean operators is essential in RasMol in order to select specific portions of the molecule. For example, if you wish to select the sidechain of histidine 63:
  - RasMol>select his63 and sidechain
This command selects the atoms that meet both criteria: being a part of histidine 63 and an atom within the sidechain (as opposed to an atom within the backbone).
- As in math, operations that are placed within parentheses are performed first.
  - RasMol>select his63 and (sidechain or alpha)
This command will select the sidechain atoms as well as the alpha carbon atom of histidine 63.

Hydrogen Bonds

- As discussed in Section II, hydrogen bonds can be added with the command “Hbonds.” Since alpha helices are stable structures in terms of buildability on the rapid prototyping machines, please note that we do not recommend placing hydrogen bonds in alpha helices. We do, however, highly recommend placing hydrogen bonds within beta sheets. The placement of these bonds within the beta sheets stabilizes the structure.
- To add hydrogen bonds specifically to the beta sheets:

Triangle Bond

- - RasMol>select sheets
  - RasMol>hbonds 225
  - RasMol>set hbonds backbone
Note: If your molecule has multiple chains, you will need to be more specific with your

command. Ie: RasMol>select *a and sheets

- You may notice that some hydrogen bonds may appear to look like “triangle bonds” in that they connect two alpha carbon atoms to create a triangle. The two amino acids will be N and N+2. For example: Amino Acid 6 and Amino Acid 8, or Amino Acid 55 and Amino Acid 57.
- These “triangle bonds” are not “real” and are distracting when we build the alpha carbon backbone models, and they do not add stability to the physical model. We recommend that you remove these bonds.
- To remove these bonds, select the amino acids and remove the bond.
  - RasMol>select 6 or 8
  - RasMol>hbond off
    - Note: if you molecule has multiple chains, you will need to be more specific with your command. Ie: RasMol>select *a and (his6 or arg8)

Adding Sidechains with a clean backbone

- If your story requires that you display specific amino acid sidechains to the model, we recommend that you do so in a way that only displays the sidechain atoms, rather than all of the atoms of the amino acid. In previous sections, we have simply selected the amino acid and displayed all of the atoms. In this section, we are going to use the Boolean operators to select just the atoms in the sidechain and display only these atoms.
- To select and display only the atoms of the sidechain of a specific amino acid:
  - RasMol>select his63 and (sidechain or alpha)
    - This command selects the amino acid (histidine 63), but limits the selected atoms to the sidechain atoms and the alpha carbon of that amino acid. It is important to select the alpha carbon atom in addition to the sidechain atoms because we need to attach the sidechain atoms to the alpha carbon. If we do not select the alpha carbon, the sidechain will build as a separate unit from the rest of the molecule
  - RasMol>wireframe 225
  - RasMol>spacefill 275
    - These two commands together will generate a ball and stick appearance. You can enter just the wireframe command and create a “sticks” appearance, but you cannot enter just the spacefill command. If you enter just the spacefill command, the atoms will be displayed as just little spheres and the spheres will not be connected to one another. It is therefore imperative to add the wireframe command in order to connect the spheres together.
- If you just select the amino acid with command of “select his63”, you will select all of the atoms within the amino acid and will generate a “bumpy backbone.” Unless the backbone atoms of the amino acid (the amino nitrogen or the carbonyl oxygen) play a specific role, then we generally do not recommend displaying these atoms on the model. It is typically (although not always) the sidechain that has the specific chemical role within the molecule and plays the key role within your story. Therefore, this should be the part that is displayed within your molecule.
- If you need to selectively display the backbone atoms and not the sidechain atoms, see number 10 for additional special design commands.

Use of Command Window versus the Pull-Down menu for designing molecules

- We recommend that you use the command line window exclusively when designing your model, rather than using the pull-down menu in combination with the command line window.
- When using the command line window, you can create your model in a step-by- step fashion and you can selectively add or subtract features.
- The pull-down menu is limited in terms of what features can be displayed. For instance, you cannot selectively display sidechains through the pull-down menu. Combining the two command features can potentially cause problems in your design work as the pull-down menu options will could potentially over-write any designs that you have created through the command line window.
- To illustrate this feature, proceed through the follow exercise:
  - Open 1A3N.pdb
  - RasMol>restrict *b
  - RasMol>backbone 300
  - RasMol>wireframe off
  - RasMol>select helices
  - RasMol>color red
  - RasMol>select his63 and (sidechain or alpha) and *b
  - RasMol>wireframe 225
  - RasMol>spacefill 275
  - RasMol>select his92 and (sidechain or alpha) and *b
  - RasMol>select *b
  - RasMol>color cpk
  - Use the pull-down menu to choose Cartoon
    - Notice how the wireframe/backbone combination disappeared and was replaced entirely by the cartoon format.
    - More importantly, notice that the sidechains that you selectively displayed are no longer present. The pull-down menu has overwritten what you have created using the command line window.
  - Return to the command line window
  - RasMol>wireframe 225
    - Notice how you added the wireframe to the cartoon, rather than replacing it.
  - Return to the command line window
  - RasMol>backbone 300
    - Notice that you have added the backbone on top of the pre-existing cartoon and wireframe format.
    - In order to remove the cartoon feature, you will need to turn this off.
  - RasMol>cartoon off
- If you alternate between the pull-down menu and the command line window, you may end up with an odd combination of features, or you may lose work that you have you created within the model. This can be very frustrating, especially if you have spent time designing your model, all to be lost within a few seconds of using a pull-down menu command.
- For this reason, we recommend that you exclusively use the command line window for your design work.

Recommended values and colors to use within your model

- During the summer of 2005, the Center for BioMolecular Modeling and 3D Molecular Designs purchased a new Z Corporation printer. This new printer is more accurate with its printing and we have developed a new guideline for recommended values for designing a model. These are the design values that we recommend using if your molecule is 200-1000 amino acids in length. If your molecule is smaller or larger, please contact us for other recommended values.
  - Backbone 300
  - Spacefill 275
  - Wireframe 225
  - Monitor lines 225
  - Hydrogen bonds 225
  - Disulfide bonds 225
- We also have recommendations with respect to colors:
  - The printer is a CMY printer (cyan, magenta, yellow)
    - Therefore, cyan, magenta and yellow are considered to be the “pure” colors and will be the brightest.
    - All other colors will be a mixture of these three colors.
    - Secondary colors created by mixing any two of the primary colors are also a good color choice. Ie: cyan and yellow will give you green.
    - We will NOT print black.
  - Colors should highlight features. In your model, the colors that “jump out at you” should be on the features that you want to emphasize in your model to assist in telling a specific story.
    - For example, you would not want your monitor lines to be colored magenta and everything else colored a light color, since the monitor lines will become the focal point of your model and the monitor lines should be the least important feature of your model. You should reserve the bright colors, like magenta, for the important features, such as the helices or the sidechains of the active or binding site.

Additional Design Commands

- Selecting a specific atom
  - If you would like to specifically select an atom, you would use your mouse to identify the atom number, (see Section 1 for further information on how to do this) and then you would use the “select” command and the “atomno=” command, which is needed to identify the atom number, rather than the amino acid number.
    - If a number is entered into the prompt line, the default assumption is that the number is the Group Number (such as the amino acid number). If you wish to specifically select the atom number, you need to designate the number as the atom number.
      - RasMol>select atomno=376

This command will select the atom within the molecule that is numbered 376.

- Selecting a region within the molecule
  - You can select a region within the molecule by selecting the range of amino acids or a range of atoms
    - Range of Amino Acids
      - If you would like to select amino acids within a range, you can do so by entering the following command:

RasMol>select #-$

The # indicates the number of the first amino acid of your range and the $ indicates number of the last amino acid of your range.

For example:

If you wished to select amino acids 4-31 of the molecule, you would enter the following command:

RasMol>select 4-31

- - - Range of Atom Numbers
Note: If you need to select 4-31 of a particular chain, you need to include that within your selection criteria.
- RasMol>select *a and (4-31)
  - If you would specifically like to select a range of atom numbers, you can do so by entering the following command:
    - RasMol>select (atomno>= ) and (atomno<= )
      - the underline region is where you would insert the specific atom number of your range
    - For example:
      - If you would like to select all of the atoms between 436 and 862:

RasMol>select (atomno>=436) and (atomno<=862)

Turning the monitor labels off
- With each monitor line added to the molecule, there will appear a number next to the line that designates the length of the monitor line in angstroms. If you are exporting this image, the numbers may clutter the image. Or, if there are several monitor lines, the numbers may hinder your viewing of the molecule. You may then wish to remove the labels from the molecule.
- To turn off the labels, enter the following command:
  - RasMol>set monitor off
- Note: Make sure that you have the word “set” included in the command. If you enter the command “monitor off”, all of the monitor lines that you have added to the molecule will be turned off.
Repeating a command
- By pressing the up arrow key on the keyboard, the previous commands that you have entered into the command line window will be repeated.
Selecting specific types of atoms
- If you wish to select a subset of atoms, such as all of the alpha carbons, you can do so by using the “*” command and the 1 or 2 letter codes for the different atoms within the molecule
- For example, if you wished to select all of the alpha carbons:
  - RasMol>select *.ca
    - The “ca” refers to the alpha carbons

Atom in Backbone	RasMol Designation
Nitrogen	n
Alpha Carbon	ca
Carbonyl carbon	c
Carbonyl oxygen	o

Conclusions

At the end of this section, you should feel comfortable designing a molecule that could successfully be built on the Z Corporation machine. To this end, you should be comfortable with the following items:

Adding hydrogen bonds
Removing triangle bonds
Adding monitor lines
Adding sidechains with a clean backbone
Using “good” colors and “good” values

Appendix to RasMol Training Guide for Macintosh Users

Mouse Control keys

As those of you who use Macintosh computers are aware, there are differences with the mouse and keyboard keys between PCs and Macs. The table below provides a comparison between the mouse/key combinations needed to perform different functions within RasMol between a PC and a Mac.

Action	Windows	Macintosh
Rotate X, Y	Left	Unmodified
Translate X, Y	Right	Command*
Rotate Z	Shift-Right	Shift-Command*
Zoom	Shift-Left	Shift
Slab Plane	Ctrl-Left	Ctrl
*On some Macs, the Option (Alt) key has the same effect on RasMol as the Command key.

Changing the PDB extension

When you download a PDB file from the Protein Data Bank, the file is saved with a .pdb

extension. Macintosh users should change the .pdb extension to .txt. For example, 1a3n.pdb becomes 1a3n.txt.

In some cases, we have had users need to add the .txt to the .pdb, rather than replacing the .pdb. In this case, 1a3n.pdb becomes 1a3n.pdb.txt.

Editing Script files

As with script files generated on a PC, script files that will be used between Mac computers will need to have the path name modified in order to be opened when moved from one computer to another (or from one computer to another). The modification of the script file is more specific with script files on a Mac than on a PC.

On the PC script file, this is how it appears: #!rasmol -script

# File: kchannel.spt

# Creator: RasMol Version 2.6 zap

load pdb “D:\CBM\Summer 2006\RasMol Training 2006\RasMol Training II\1J95.pdb”

background [255,255,255]

set ambient 40 set specular off

On a script file that was generated on a Mac, this is what will appear: #!rasmol -script

# File: kchannel.spt

# Creator: RasMol Version 2.6

zap

load pdb “MacIntosh HD:Users:SColton:Desktop:Kchannel:1J95.txt” background [255,255,255]

set ambient 40 set specular off

Notice that there are colons, rather than back slashes. Notice that the computer username is listed after users.

Notice that the location of the file (desktop in this case) is listed.

Notice that the folder in which the PDB is located is listed (Kchannel in this case). Notice that there is a .txt extension, rather than a .pdb extension.

Please NOTE: If you move the script file from a Macintosh computer to a PC computer, it will be essential to change the load command line to the PC format (with the back slashes, rather than the colons). Likewise, if you move a script file from a PC to the Macintosh computer, it will important to change the load command line to the Macintosh format.

Operating Systems

If you have OS X on your computer, in order to run RasMol, you have to have Classic Environment (emulator) installed on your computer. If you need to install this on your computer, you can visit http://docs.info.apple.com/article.html?artnum=151871.

Overview of The Tutorial

This tutorial gives you an opportunity to learn how to explore the structure and action of biomolecules using computer graphics. You will learn how to obtain macromolecular structure files from the Protein Data Bank, and to display and manipulate molecular structures using the program RasMol. With the skills you learn here, you can independently examine any macromolecular structure available. Many structural biology sites on the World Wide Web provide structures, illustrations, and even animations that you can view with RasMol. You can obtain RasMol free for use on your own computer from Rasmol

Have RasMol installed on your computer (but first read below about Conventions used in the instructions of this tutorial).

RasMol for the Macintosh is called RasMac, but I refer to the program as RasMol throughout the tutorial. I wrote the tutorial using RasMac 2.6b2 for Macintosh. Most of the instructions should be the same for Windows versions of RasMol. Where I am aware of differences between Mac and PC versions of the program, I added notes in parentheses for PC users.

The name RasMol comes from raster display of molecules. Raster is a type of computer display especially useful for showing solid surfaces. It may not be a coincidence that the letters Ras are also the initials of RasMol’s creator, Roger A. Sayle of Glaxo Corporation and the University of Edinburgh, Scotland. Sayle began developing this program as part of this graduate work in computer science, and continues to expand it with support from Glaxo. Thanks to Roger A. Sayle and Glaxo for giving this powerful tool to the world structural biology community.

Conventions

In this tutorial, instructions for giving commands or using menus will appear in a consistent format. Here are some sample instructions and their meanings:

< return > (keyboard entry)

means press the key labeled return on the computer keyboard. All key-press instructions are surrounded by < >’s.

Display: Backbone (menu command)

means pull down the Display menu and select Backbone. All menu instructions are in bold type

with a colon between menu name and command name.

RasMol > select alpha < return > (command on RasMol Command Line)

means that you should type the command “select alpha” and then press the returnkey. Any words that you type while RasMol is running appear in the Command Line window beside the RasMol prompt: “RasMol >.” When you type the command and press return, RasMol executes the command.

button: Open (on dialog windows)

means that you should click once on the button labeled Open.

link: Other Sites of Interest (on web pages)

means that you should find and click the link, usually a word or phrase in colored text, that says, “Other Sites of Interest”.

Filenames are in italics — for example, 3b5c.pdb.

Getting Started

NOTE: Plan to work at least through section 4 of this tutorial without stopping. Thereafter, the end of any section is a convenient stopping point. See the end of section 4 for instructions on how to resume the tutorial after stopping.

RasMol works better with Netscape if it is located on the desktop. Make a copy of version 2.6b2 of RasMol (for PC) or RasMac (for Macintosh) and the file rasmol.hlp on your desktop.

Configure your web browser to use RasMol or RasMac (hereafter referred to as RasMol) as a helper application for files of MIME type chemical- x/pdb, with suffix .pdb. For help with this task, see Configuring Netscape.

Use your web browser to obtain the atomic coordinate file (or PDB file) with code 3b5c from the Protein Data Bank. For help with this task, see Getting PDB Files. Place the file on your desktop and name it 3b5c.pdb. {/usr2/tutorials/pdb/3b5c.pdb}

The web page PDB File Contents provides a complete description of the contents of a PDB file, thus allowing you to learn what information RasMol uses to create molecular graphics displays. But first, get started with viewing. After you have learned some basics, the contents of the PDB file will mean more to you.

Windows and Help

Start RasMol by dragging the file icon of 3b5c.pdb onto the RasMol icon (UNIX: type rasmol at the unix prompt). After the file is loaded, two windows appear. RasMol’s Main Window displays a wireframe model of cytochrome b5. Behind the Main Window (UNIX: in the unix shell window) is the Command Line window, which you use to issue commands to RasMol. (If you are working in Windows, the Command Line window starts out minimized to an icon at the bottom of the screen. If you don’t see it, hold down the Alt key while you press the Tab key repeatedly.

When the banner says “RasMol Command Line”, let up both keys and the Command Line window will open.) When RasMol needs to tell you something, its messages also appear in the Command Line window. When RasMol is waiting for a command, the prompt,

RasMol >

appears on the command line, which is the last line in the window. Arrange the windows conveniently, as follows:

Windows: Command Line

The Command Line window comes to the front. You can also accomplish this by simply clicking on any exposed part of the window. Grab the window, by placing the mouse pointer on its top border and holding down the mouse key, and drag it to move it. Place it so that its lower left corner is very close to the lower left corner of the screen.

< return >

Each time you press return, the cursor moves down the window and prints the prompt, “Rasmol

>” on the command line. Keep pressing return until the prompt appears at the very bottom of the window. Now use your first command to get a brief introduction to the program:

RasMol > help < return > Read the introduction.

RasMol > help commands < return >

This information includes a list of the most frequently used RasMol commands. Any time you

would like to know more about a command, just type help followed by the command.

Windows: Main Window

The Main Window returns to the front. You can also accomplish this by simply clicking on any exposed part of the Main Window. Click on the small square at the top right of the window. The window expands to fill the screen. Then drag the small square at the bottom right of the window straight upward to shrink the window just enough to expose the last two lines of the Command Line window. With this arrangement, the Main, or graphics, window is as large as possible, but you can also see the command line and RasMol’s messages.

In the Main Window, you are now seeing a wireframe model of the protein cytochrome b5, a protein involved in redox reactions in the liver. This small protein contains 86 amino acid residues and a heme prosthetic group with a central iron ion that is the site of oxidation or reduction. The structure you are seeing is the oxidized (Fe³⁺ or iron [III]) form.

When RasMol starts, it always shows a wireframe model of all the atoms in the protein and in any other molecules (such as cofactors or inhibitors) that are included in the PDB file.

Now you will simplify the display and learn how to manipulate the model.

Rendering

Display: Backbone

Now you see only the alpha carbons of the protein, connected by straight lines. Lines connecting alpha carbons are sometimes called “virtual bonds.” You can begin to see secondary structural elements such as alpha helices and strands of beta pleated sheet. Try some of the other Display commands. Each command in the Display menu changes the way RasMol represents, or renders, the model. End by displaying the backbone model. Now you will make the secondary structural features even more obvious.

Colours: Structure

This command colors alpha helices hot pink, beta strands yellow, beta turns blue, and other parts of the model light gray (not my choice of colors!).

RasMol > select hetero and not hoh < return >

This command selects only the hetero (non-protein) groups in the file, excluding the water molecules that are frequently included in crystallographic models. Nothing happens until you issue another command. Commands only affect currently selected atoms. Also notice that you do not need to bring the Command Line window to the front in order to type commands.

Display: Ball & Stick

RasMol displays a ball-and-stick model of the selected atoms. Notice that this command does not affect the protein chain, because it is not selected.

Colours: CPK

RasMol colors the selected atoms according to widely used chemical conventions: carbon is light gray, nitrogen is light blue, oxygen is red. Notice again that the menu command affects only the currently selected atoms.

Click on any alpha carbon of the protein and watch the command line. This is called picking an atom. When you click on an atom in the Main Window, RasMol identifies it. You should see something like this:

Atom: CA 652 Group: PRO 81

This tells you that you clicked on the alpha carbon (CA) of residue 81 in cytochrome b5, which is a proline. RasMol calls a residue a group, and a nonprotein portion of the molecule, including a water molecule, a hetero. If you clicked on some other atom, use picking to find this one.

If you can see a yellow atom in the space filling model, click on it. You should see

Atom: FE 754 Hetero: HEM 201

The yellow atom FE is iron (Fe³⁺, actually) in the center of the heme group of cytochrome b5. By using the command select hetero and not hoh, you can quickly find nonprotein groups in the PDB file. By picking any atom in the group, you can learn the PDB name of the group, and thus know how to specify it in commands.

Note: Picking works best with Wireframe and Sticks displays, does not work at all with

Ribbons, Strands, or Cartoons, and is somewhat unpredictable with Spacefill and Ball&Stick. This is not a great inconvenience, because Wireframe is the most useful display for exploring unfamiliar models in detail.

Listed below is the sequence of commands you have used so far. You can use this sequence to give you a quick overview of any macromolecule for which you have a PDB file, and to reveal any hetero groups (cofactors, inhibitors, water, and so forth) that are present in the file. The sequence is

Display: Backbone Colours: Structure

Rasmol > select hetero and not hoh < return >

Display: Ball & Stick Colours: CPK

Now you will learn how to manipulate the model. You can rotate it, move (translate) it to a different part of the screen, and zoom in or out.

Manipulations

To rotate, place the pointer anywhere on the Main Screen, hold down the mouse button and move the mouse. This is called dragging the pointer (as opposed to moving the pointer without holding down the mouse button). Dragging up and down rotates the model around a horizontal (x) axis through its center. Dragging left and right rotates around the vertical (y) axis. Hold down shift and command (UNIX: shift + right_mouse) keys while dragging left and right to rotate the model around the z-axis, which is perpendicular to the screen.

To move the model around on the screen (called translation), hold down option (UNIX:

right_mouse) and drag. The model moves across the screen, following your mouse motion. To translate or to rotate about the z-axis, you don’t even have to hold down the mouse button

To zoom, hold down shift and drag the pointer down the screen to bring the model closer, or up to move it farther away.

These mouse motions allow you to move the model around and orient it so that you can see its features clearly. Such motions are a fundamental part of any molecular modeling program. Take time to play with the the model by rotation, translation, and zooming. You can always return to the original orientation like this:

RasMol > reset < return >

You will often need to rotate, translate, or zoom the model in order to carry out the instructions that follow. Movement helps you to see the molecule better, especially to distinguish near from far.

You may conveniently take a break from this tutorial at the end of this or any later section. To resume your work at the beginning of any section, start the desktop copy of RasMol and open 3b5c.pdb. Arrange the windows as in section 1.

Picking, Selecting, and Restricting

Use picking to find residues 64 and 72. You may need to manipulate the model to bring these residues into view. Notice that residues 64 through 72 constitute an alpha helix. Now you will examine the helix more closely.

Rasmol > restrict 64-72 < return > The restrict command selects all atoms specified, and

removes all others. It does not alter the display of the selected atoms. Subsequent commands will affect only the selected atoms.

Display: Sticks Colours: CPK

RasMol draws residues 64 through 72 as a stick model. Try rotating the model so that all main chain carbonyls point upward. You will find that the rotation behavior is strange, because RasMol is still rotating about the center of the whole molecule. Fix this as follows:

RasMol > set picking center < return >

Now find the lysine residue at one end of the helix. Click on its alpha carbon. The command line should read

Rotating about lys72.ca (578)

If some other atom is listed, search again for the alpha carbon of lysine 72 and click on it to get this message. RasMol will now rotate the model around this atom.

RasMol > set picking ident < return >

This resets picking to identify picked atoms without changing the centering of rotation. After issuing a command, always check the command line to see if RasMol gives back a message. If there is no message, it means that RasMol recognized the command. RasMol reports syntax errors in commands if it cannot recognize them.

Now you will reduce the display further to just one residue.

RasMol > restrict 72 < return >

Now only the lysine residue is shown. Bring the residue to the center of the display. Click on the atoms of this one residue, starting with the alpha nitrogen (blue, near the red oxygen), then the alpha carbon attached to it, the carbonyl carbon, and the carbonyl oxygen (red). Note their atom names on the command line. In the same order, they are N, CA, C, and O. In the PDB file, these are the names of the main chain atoms of each residue. Now find the atoms of the side chain and click on them, noting their names on the command line. Starting next to the alpha carbon, they are the beta carbon (CB), gamma carbon (CG), and so forth, out to the zeta atom, which is nitrogen (NZ). PDB files contain lists of all atoms in a protein, named in the same way as this residue, along with the data needed to display them as a graphics model.

In addition to display data, a PDB file contains other useful information. This is a good time for you to take a break and look at PDB File Contents.

Now you will bring the entire alpha helix back into the display, and center the rotation about the alpha carbon of the central residue, arginine 68.

RasMol > select 64-72 < return >

Display: Sticks

RasMol > center arg68.ca < return >

The last command is another way to change the center of rotation. Notice the syntax of the atom expression that follows the word center. An atom expression describes a particular atom or set of atoms. This expression for a single atom consists of the abbreviation of the residue (arg), followed — without a space between — by the residue number (68), a period, and the atom name (ca for alpha carbon). An atom name is always preceded by a period. The previous command also contained an atom expression, 64-72. RasMol is pretty picky about the syntax of these expressions, so take careful note when you encounter them. You can use these expressions in select and restrict commands as well.

For more information about expressions, type help expressions, help primitives, and help examples. Although atom expressions may seem daunting to you now, you will see many examples before you complete the tutorial.

Arrange the helix so that it takes up most of the screen, with carbonyl carbons pointing upward. Press the cursor-up key next to the keypad and watch the command line. Each time you press cursor-up, RasMol reprints your previous command. Keep pressing until you bring your restrict 64-72 command back to the command line. If you go past it, use cursor-down to retrieve more recent commands. When the command is on the command line, add and mainchain to the command, and press return. In other words,

RasMol > restrict 64-72 and mainchain < return >

The side chains disappear, giving you a clear view of the main chain conformation in an alpha helix. You have restricted the selected atoms to residues 64 through 72, and to the mainchain atoms of the protein. Mainchain is one of many sets that you can specify in RasMol commands. The mainchain set includes only atoms N, CA, C, and O from each residue. For a listing of the names of other sets, type help sets return.

RasMol > hbonds < return >

RasMol draws dotted lines for all the main-chain hydrogen bonds of currently selected residues. Now use picking to confirm that most hydrogen bonds in an alpha helix are from the carbonyl

oxygen of residue n to the N-H of residue n + 4. Note that hydrogen atoms are not present in this file. In the x-ray crystallographic image of proteins, it is usually not possible to resolve the hydrogen atoms, but we deduce their locations from principles of structural chemistry.

RasMol only looks for hydrogen bonds between mainchain atoms, such as those in helix and sheet. What is more, it “finds” the bonds by simple criteria of geometry and distance, criteria that are sometimes met more than once by the same atom (for instance, notice the two hydrogen bonds drawn to the nitrogen of lysine 72). In this case, the real bonding situation is unclear.

Viewing in Stereo

(If you are resuming the tutorial after a break, start your desktop copy of RasMol, arrange the windows as in section 1, and open 3b5c.pdb. Restrict the view to residues 64-72, mainchain only. Turn on hydrogen bonds.)

Options: Stereo

RasMol displays the model as a stereo pair. Viewed properly, a stereo pair gives you a three- dimensional (3D) image of the model. Take time now to begin learning this skill. It takes some practice, and you may find it slightly uncomfortable at first, but it will become easy and comfortable, and your effort will be richly rewarded by increased power to see spatial relationships and structural details that are much harder to see any other way.

Here’s how to view in 3D. Gaze at the screen, keeping your head level (don’t tilt it left or right), and cross your eyes slightly. As you know, crossing your eyes makes you see double, so you will see four images. (By the way, you can’t hurt your eyes or eye muscles by crossing your eyes, and you can’t get them stuck that way.) Try to cross your eyes slowly, so that the two images in the center come together. When they converge or fuse, you will see them as a single 3D image. The fused image will appear to lie between two flat images, which you should ignore. When you are viewing correctly, you see three images instead of four. The center image is three-dimensional.

At first, the 3D image may be blurred. Keep trying to hold the stereo pair together while you focus. The longer you can hold it, the more time your eyes have to adjust their focus. Usually, even before you begin to get the hang of focusing, the two central images lock together, because your mind begins to interpret them as a single 3D object.

Having trouble? Here’s another approach. With your head level and about 2.5 feet from the screen, hold up a finger, with its tip about 6 inches in front of your face, and centered between the stereo pair on the screen. Focus on your finger tip. Without focusing on the screen, notice how many images you see there (they will be blurred). If you see four images, move your finger slowly toward or away from you eyes, keeping focused on your finger tip, until the middle pair of images converge. With your finger still in place, partly covering the converged pair, change your focus to the screen. The image partly hidden by your finger should appear three- dimensional. Your finger should still appear single, but blurred. With some practice, you can remove your finger and still keep the screen images converged into a stereo image.

Too often, people try only briefly and halfheartedly to view in stereo, and never try again. Almost anyone can view in stereo with a little effort and practice. The only ones who simply cannot are those who have acute amblyopia (one very weak eye). And those who say they can see just as much without stereo simply cannot imagine what they are missing. You can continue this tutorial with or without stereo viewing, but if you ever need to explore macromolecules on your own, you can be a much more effective explorer if you learn to see in 3D.

(This viewing method is different from that needed for viewing printed stereo pairs in textbooks and journals. To view most printed images, you need to view the left image with the left eye, and the right image with the right eye (called divergentviewing). In addition, with printed views, the distance between the images must be less than the distance between your eyes, so the images must be small. Viewers are available to magnify the image and to guide your eyes. To view larger stereo pairs, such as those on a computer or projection screen, you must use the method described above, and cross your eyes slightly to look at the right hand image with your left eye, and at the left-hand image with your right eye (called convergentviewing). For most people, convergent viewing is easier to learn, but most structural biologists learn to view both ways without viewers.

One unfortunate quirk of the current version of RasMol is that picking is sometimes unpredictable in stereo. You should always pick on the left image, but if you do not get proper response to picking, turn off stereo for picking by selecting Options: Stereo. (The check mark appears by the word Stereo when the stereo display function is turned on, and disappears when it is turned off — this type of on/off command is sometimes called a toggle.)

Exploring

Display: Spacefill

Rotate this model to view it end-on. Notice that stick models make protein structures appear very open and empty, but even an isolated helix is quite densely packed.

RasMol > restrict 64-72 < return > RasMol > hbonds off < return > Display: Spacefill

Now you see a space filling model of the entire helix. The side chains reappear because this

restrict command includes them.

RasMol > select < return > Now all atoms are selected.

Display: Backbone Colours: Structure RasMol > reset < return >

This returns the full backbone to the screen, centers rotation on the center of the whole model, and returns it to the original orientation. The last four commands are useful when you get lost and need to redisplay everything and get your bearings again.

RasMol > restrict sheet < return >

This command removes all residues except those in pleated sheet (sheet is another set). Center rotation on one of the alpha carbons in the middle of the sheet (look back at previous commands if you don’t remember how, and remember to set picking back to ident after you change the center of rotation). Display the model as sticks and color it CPK. Display hydrogen bonds. To help you see the sheet structure more clearly, remove the sidechains as follows:

RasMol > restrict sheet and mainchain < return >

Decide whether the three central strands in the sheet are parallel or antiparallel. What about the edge strands? Are they parallel or antiparallel to their neighbors? Here’s how to check your answers:

Display: Cartoons

Cartoon displays show sheet strands as arrows pointing toward the C-terminal end of the chain. A pair of chains with arrows at the same end are parallel. If arrows on neighboring strands are at opposite ends, the strands are antiparallel.

RasMol > select < return > RasMol > hbonds off < return > Display: Cartoons

Colours: Structure

Now you see the whole protein as a cartoon. This is a vivid display that is easy to interpret, but it has one disadvantage for further exploration: picking doesn’t identify atoms. Remember that you must us a display function that shows the exact location of atoms in order to identify atoms by picking.

RasMol > restrict turn < return >

Display: Backbone

Now you see the beta turns in the model.

RasMol > restrict 17-21 < return >

This shows just one turn. Display it as sticks in CPK colors, and turn on hydrogen bonds. Notice that residue 19, the middle residue, is lysine.

RasMol > center lys19.ca < return >

Confirm that the hydrogen bond in a beta turn connects the carbonyl oxygen of residue n with the N-H of residue n+3. From information in a standard biochemistry textbook, decide whether this is a type I or type II turn. One way to look at it: if the three carbonyls that make the turn (residues 17, 18, and 19) all point in the same general direction (up or down when you look at the turn edgewise), then it the turn is type I. If the middle carbonyl points in the opposite direction from the other two, it’s type II.

RasMol > reset < return > RasMol > select < return > RasMol > hbonds off < return > Display: Cartoons

Colours: Structure

RasMol > select hetero and not hoh <enter>

Display: Spacefill Colours: CPK

Now you see the heme prosthetic group in its cartoon-protein binding site. You will now use RasMol to explore the binding of the heme to the protein.

Rotate the molecule so that you see the heme edge-on, protruding from the right- hand side of the protein. Notice that the binding pocket is composed of four alpha helices above and below the heme, and a four-strand pleated sheet on its inside edge. The molecule looks somewhat like a pair of jaws holding a heme. The teeth are four alpha helices, and the throat is pleated sheet.

However, this cartoon view does not display any of the chemical details of heme binding.

RasMol > restrict within (7.0, hem) < return >

Display: Wireframe Colours: CPK

RasMol > center hem.fe < return > RasMol > select hem < return > Display: Ball & Stick

Now you see the heme as a ball and stick model surrounded only by atoms that lie within 7.0 angstroms of heme atoms. The restrict within and select within commands are powerful tools for directing your attention to specific interactions within macromolecules.

Look for possible electrostatic interactions and hydrogen bonds between the heme (ball & stick) and the protein (wireframe). First, look at the yellow iron (III) ion in the center of the heme. The ion is part of an octahedral transition metal complex. Four of its six ligands are the light blue nitrogens of the heme porphyrin ring. What are the other two ligands? Use picking to identify them. When you have learned the residue numbers of the side chains that contain the ligands, display them in stick form as follows:

RasMol > select (#1 or #2) and sidechain < return >

(substitute the residue numbers of the ligands for “#1” and “#2”).

Display: Sticks

Now you should see two histidine side chains providing nitrogens as the fifth and sixth ligands of iron(III). The stick display does not include the alpha carbon in the main chain, because the alpha carbon is not included in the selection sidechain, which selects CB but not CA. You can, however, include CA in the display, and complete the stick model of the histidine side chains without changing the selection, as follows:

RasMol > set bondmode or < return >

Display: Sticks

Notice that one bond, the CA-CB bond, is added to the display for each histidine. RasMol has two bond modes, called and and or. In bond mode and, RasMol draws bond CA-CB if both CA and CB are selected. In bond mode or, RasMol draws bond CA-CB if either CA or CB is selected. When you start RasMol, the bond mode is and until you change it. Consider your last two sticks commands, with CB selected and CA unselected. On the first try, in the and bond mode, RasMol did not draw the the CA-CB bond as a stick because CB was selected and CA was not. Then on the second try, in the or mode, RasMol drew the CA-CB bond as a stick because CB was selected. Changing the bond mode does not change the current display, but it does change the behavior of subsequent display commands.

RasMol > set picking monitors < return >

Working without stereo, click first on the iron (III) ion, and then on one of its histidine nitrogen ligands. A dotted line appears between the atoms, along with a label showing the distance between the atoms in angstroms. The label is colored that same as the first atom picked. If you accidentally pick the wrong atoms, you can remove the dotted line and label by picking the same two atoms again. Now measure the distance between iron (III) and the other histidine ligand.

This distance, about 2.0 anstroms, is the length of the bond between iron (III) and nitrogen in the transition-metal complex. Such bonds were once called coordinate-covalent bonds, because one of the two bonded atoms, in this case the ligand nitrogen, donates both of the electrons to form the bond.

The heme has two chains that extend from the edge that sticks out of the binding pocket. Both chains end with carboxyl groups. Can you find any heme-protein interactions that involve the

heme carboxyls? There are two hydrogen bonds involving serine 64. Find and measure them. To make all your measurements easier to see, use the cursor-up key to retrieve and execute your command restrict within (7.0, hem). Then display all the atoms of the heme and its binding pocket in wireframe. Another quirk of RasMol is that the heme iron is not displayed in wireframe. Select only the iron (hem.fe) it and display it as a ball using Ball & Stick.

Now you will study the remaining interactions that hold the heme in place, which are primarily hydrophobic.

RasMol > set picking ident < return > RasMol > monitors off < return >

This sequence resets picking to the default and removes all measurement lines and labels.

RasMol > select hydrophobic and sidechain < return > RasMol > color yellow < return >

This sequence selects only the sidechains of hydrophobic residues, and colors them yellow. The hydrophobic set is another set of atoms you can use in select commands. The color command does not add atoms to, or remove atoms from, the display. To see the names of other colors you can use in commands, look at help for color and colors. (In the colors help information, RGB stands for red- green-blue.)

RasMol > select hem.c?? < return > RasMol > color cyan < return >

This sequence selects all carbons in the hem and colors them cyan. The question marks stand for unspecified characters. The selection hem.c?? means, “atoms of heme designated c followed by up to two additional unspecified characters.” Now you will display the heme and its binding pocket as a space filling model.

RasMol > restrict within (7.0, hem) and not hoh < return >

Arrange the model so that you are looking into the opening of the hem pocket, with the two heme carboxyls pointing at you. It is much easier to do it stereo. If you can’t tell front from back, try displaying in ball and stick, which gives more depth cues.

Display: Spacefill

Now you see the heme peeking out of its pocket. One carboxyl points out into space, and the other is in contact with two atoms of serine 64. The cyan carbons of the heme are hydrophobic, as are the yellow carbons of the hydrophobic sidechains. In this view, you see hydrophobic interactions, therefore, as contact between cyan and yellow. But much of the contact area is buried by the space-filling models. Let’s look inside.

RasMol > slab 100 < return >

Now hold down the control key at the bottom left of the keyboard (not the command key). While holding the key down, move the mouse pointer up the screen. As you move the mouse, an invisible plane slides back through the model, cutting away everything that lies in front of it.

Thus you can slice into the model to any depth, removing all foreground as you go. Slide the pointer up and down the screen to change the position of this cutting plane. (You may find that the action is choppy, because the computer is doing many calculations to produce each successive view.) As you cut into the model, notice the contacts between heme carbons (cyan) and atoms of hydrophobic side chains (yellow). By releasing the control key, you can rotate the model to cut into it from other directions.

RasMol > slab off < return >

Again orient the model so you are looking at the heme peeking out of its pocket.

RasMol > select hem < return >

Display: Wireframe

RasMol > select hem.fe < return >

Display: Ball&Stick

Now you can clearly see the interior of the pocket, and observe its strongly hydrophobic nature. You can also see the two histidine side chains that protrude into the pocket to interact with the iron (III).

RasMol > select hem < return > RasMol > dots 200 < return >

This display colors the surface of the heme with about 200 dots per atom. Dot displays give a feeling of solidity, but you can see through them to neighboring atoms. Zoom in and try to see contacts between heme carbons and other atoms in the pocket. These contacts are much easier to see in stereo. For a dramatic view of the pocket, try turning the model around and slabbing in through the back. You will see what the world looks like from the perspective of the iron (III) ion.

File: Close

Cytochrome b5 is a small protein consisting of a heme and only one polypeptide chain. In your biochemistry class, you will also study oligomeric proteins, which consist of more than one polypeptide subunit, as well as protein-protein and protein- nucleic acid complexes. RasMol has some features that are very useful with models that contain more than one chain. Now you will briefly examine such a model — part of an antigen-antibody complex.

Oligomeric Proteins

Obtain the PDB file with code 2iff (see section 1 for a reminder about how to get a PDB file). After the file appears on the desktop, rename it 2iff.pdb. Then drag it onto the RasMac v2.6 icon to open it. For sections 8 and 9 of this tutorial, you must make sure that you are using the desktop copy of RasMol; otherwise you may have difficulty finding files that you create using RasMol.

The file 2iff.pdb is larger than 3b5c.pdb and takes longer to load. When loading is complete, RasMol provides some information about the file in the Command Window, and shows a complete wireframe model in the Main Window. Arrange the windows as described in section 1. Move the model around to get some feeling for its size and complexity in comparison to cytochrome b5.

Display: Backbone Colours: Chain

RasMol presents each chain in a different color. This allow you to see immediately how many different chains are present. Click on any atom in the pea-green chain. (the smaller of the two chains shown in shades of green). On the command line, RasMol displays something like

Atom: CA 2574 Group: GLY 117 Chain: Y

This tells you that the green chain is designated Y in the PDB file. With oligomeric proteins, you need to know chain designation in order to compose specific select and restrict commands.

Other than the need for chain designations, exploring a multi-chain model is just like exploring a single chain. Identify the other chains by clicking on them. Chain Y is the enzyme lysozyme from hen egg white. Chains H and L are the antigen-binding parts of the heavy chain and light chain of a mouse antibody. Together, they are called an Fab fragment. The antibody was made by immunizing a mouse with hen lysozyme and then purifying a specific antibody to lysozyme So in this model, lysozyme is the antigen.

RasMol > select helix or sheet < return >

Display: Cartoons Colours: Structure

The first command selects all residues that are in either alpha helical or beta pleated sheet conformations. (The command select helix and sheet selects nothing. Why?) The Display and Colours commands affect only the selected residues, so the non-helical and non-sheet portions remain in colors that allow you to distinguish them as separate chains. You can see that the antigen (Y) contains several alpha helices and a 3-strand pleated sheet, and that the antibody chains are primarily beta structure. The two antibody chains each exhibit two domains of the immunoglobulin fold, a beta structure that is found in all antibodies, as well as some other proteins whose function is recognition.

RasMol > select :H or :L < return >

Display: Backbone Colours: Chain

RasMol > select within (6.5, :Y) and not :Y < return > RasMol > color white < return >

Parts of the antibody backbone that are close enough to the antigen to be involved in the antigen- antibody interaction are now shown in white. With a few commands, you have focused on what are called the complementarity determining regions(CDRs) of the antibody. By restricting your display to these regions and adding all atoms in wireframe, you could explore in detail the interactions that bind this antibody to its antigen.

Saving Your Work: Scripts

During detailed exploration of an unfamiliar model, you my want to put your work aside, and come back later to start with your current view. Sometimes you can invest much time and thought in producing a particularly revealing view of a model. RasMol provides a way to preserve your work. This is a great feature for illustrating lectures, because you can make vivid classroom views and bring them to the screen immediately. At any time during operation of RasMol, you can write a script that RasMol can use to recover the current view. You will now write a script.

First, rotate, translate, and zoom your model into a position that shows the CDR’s clearly, and that fills the screen nicely.

RasMol > write script CDRs.spt < return >

After a brief pause, RasMol writes the script. This new file will appear in the same location as the RasMol program icon. If you have been working, as instructed, with RasMol on your Macintosh desktop, the file will appear there. Its icon is a little scroll. The file ending .spt (for

script) helps to distinguish a script file from a coordinate file, which usually ends in .pdb or .ent. After one more exercise, you will try out your script.

Look back at the commands you have used with this file, and notice the syntax of atom expressions that designate the chain. Observe that just as .ca means atom CA(an alpha carbon),

:Y means chain Y (if you prefer, you can use *Y). In a select or restrict command, you must specify the chain unless you want the command to apply to all chains. As an example of the select syntax, select gly40:L.ca selects one atom: the alpha carbon of glycine 40 in the L chain. To test yourself on this syntax, try this: restrict the display to aspartic acid 48 of the antigen chain, display it as ball and stick, center on the alpha carbon, select the gamma carbon of this residue, and color it green.

Next, exit from RasMol.

File: Quit

This commands stops RasMol, and its windows disappear, revealing the desktop.

Find your script file (CDRs.spt) and double click on it. RasMol appears, and after a pause (perhaps a long pause!), presents the same view you were displaying when you wrote the script file.

There are some restrictions to your use of the RasMol scripts. In their original form, they can only be used from the same location (in this case, the desktop) into which RasMol saved them. This means that RasMol, the PDB file, and the script must all be exactly where they were at the time the script was prepared. Fortunately, by editing the scripts in a word processor, they can be modified and made portable. In the exercise at the end of this tutorial, you will make and save several scripts, and then transfer them to a disk to hand in. I will be able to modify them so I can see your results. If you want to use scripts yourself see ##Making Scripts Portable.

Summary

In working through this tutorial, you have used many RasMol commands and features, but there are many more available.

Exercise 1

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.

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.

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:

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:

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.

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.

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	Alanine
the groups around the chiral centre (the C-alpha atom) in this L-amino	Cysteine
acid.	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.

A set of script commands to demonstrate selections with a view of myoglobin in the 1A6M structure.

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

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
*hemp.fe**	Iron atoms in the Heme of chain P
.;A	Atoms in alternate conformation A

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.

Script to focus on the heme group of myoglobin.

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

*/4	All atoms in model 4 – this refers to multiple NMR-structure models in a single PDB file.

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.

H-bonds of the Myoglobin backbone.

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]

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 !

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.

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

SURFACES AND SLABS

Rasmol can display van der Waals and solvent accessible surfaces and color them according to properties. Try the following for illustration:

The Van der Waals surface of the heme prosthetic group of myoglobin.

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

WEB RESOURCES

A collection of useful resources for RasMol on the Web.

A “slab” or clipping plane allows you to see cross-sections of a molecule. Try this view:

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.

A cross-section of Myoglobin shows the (red) heme-group packed into the hydrophobic interior (yellow).

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

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.

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.

Exercise 2

RasMol Exercise

(1) goto PDB link to download:
1ZNI (porcine insulin)
1LPH (engineered human insulin)

(2) Double click on the Raswin icon on your computer desktop. This should call up the RasMol windows (the RasMol Command Line window will appear minimized).

(3) Click on the minimized RasMol Command Line to display the command line window.

(4) Type: background white, followed by a return. This changes the color of the background from black to white.

(5) You can rearrange and size the windows to suit you.

(6) Drag PDB file – 1ZNI and drop down to the rasmol window. You now should see the structure of insulin displayed as a wireframe model in the main window! (look also for command window.)
Questions

How many polypeptide chains (type the follow commands in the command window)

               reset
               restrict backbone
               ribbons on
               wireframe off
               color chain
One can also save a script to your floppy disk by typing: write script A:\zni.sp
               (or whichever directory that you want)
How many amino acids in chain A?

select *.ca and *a (select all CA atom on chain A)
(answer given in the command window: “21 atoms selected!”, so there are
21 amino acids in chain A)
That is N/C- terminus?

select 1,21 and *.ca and *a
label
(9) There are four polypeptide chains in this structure of insulin, each shown with a different
color. A PDB file uses a unique letter for each chain, beginning with “A”, which RasMol
recognizes. For example, try the RasMol command restrict *a. This displays only the “A”
chain in the RasMol view window. How could you use RasMol to identify the N- and
C-terminal residues of the “A” chain?

(by click both ends and see what is in the command window)
10.Is there more than one type of helix in the “A” chain?
select 13-16:a and *.ca
spacefill 200

select 16-19 and *.ca and *.a
spacefill 200

then check how many amino acid per turn.
11 What is the secondary structure of chain B?
12 .How many amino acids are in each chain? Do any of the chains have the same amino acid
sequence? The PDBSum link for 9ins has the answer.

http://www/biochem.ucl.ac.uk/bsm/pdbsum/1zni/main.html
(Please explore this site further after the class)

(13) Explore the various pull-down menus to change the presentation of the structure, under Display, Colors, and Options.

(14) Explore the functions of the left mouse button by clicking on the structure, and by dragging the mouse. Try dragging the left mouse button with the shift key held down. Try the same but with the right mouse button held down.

(15) type select HOH. Then display as ball and stick. What do you see? Display as spacefill, then as wireframe. What do you see?
(16) Show Those Disulfides

Now let’s extend our discussion. Insulin is secreted by the
pancreas into the blood stream. Secreted proteins often contain
disulfide bonds (-S-S-) due to an oxidizing environment. They are
formed when two cysteine side chains (-CH2SH) come within
about 0.3 nm (3 Å) of each other. Let’s use RasMol to see if the
“AB” monomer contains any disulfide bonds. We can do a quick
test of this by using the following commands:

reset
              restrict *a,*b
              ssbonds on
              ssbonds 100
              color ssbonds yellow

Each amino acid in a protein chain is numbered in sequence,
beginning with the N-terminal position. Identify the cysteine residues,
by number and chain, that are involved in disulfide bond formation.

(17) You learned that the AB insulin monomer has three
disulfide bonds. Let’s do a better job of displaying them:

reset
             set bondmode or
            select sidechain and cys and (*a,*b)
              wireframe 70
              spacefill 200
             set specular on
Instead of seeing just S-S bonds, we can now see the entire
cystine side chain (-C-S-S-C-), without hydrogens, and its link to
the protein backbone. Two of the helices in the 21 residue A
chain are crosslinked to the B chain. In addition, the A chain has
an intramolecular crosslink. The “CD” monomer is structurally
similar to the AB monomer. There is, however, a significant
structural difference in the B and D chains, despite identical
primary structures.

(18) What’s holding those monomers together? (1zni vs. 1lph)
The monomer is only part of insulin’s story. In the presence of
various stabilizing ions, such as Zn2+ and Cl-, insulin monomers
reversibly self-associate into dimers and hexamers.
The monomer is the active form in the blood stream because it
can interact with insulin receptor protein. The aggregate forms
(dimer, hexamer) are inactive but are physically more stable and
are therefore used in pharmacological preparations for diabetic
patients.
(The physical instability of monomers in pharmacological
preparations is characterized by partial unfolding (loss of tertiary
structure). The unfolding exposes hydrophobic surfaces that
induce long insulin fibrils to form. These fibrils do not reform
monomers and also elicit an immune response – insulin
antibodies are formed which are potentially dangerous).

The insulin response time in vivo depends on the rate at which
hexamers and dimers dissociate into active monomers following
injection into the blood stream. The stabilizing ions (zinc and
chloride) dissociate upon injection, which causes the equilibrium
to shift toward monomers. The rate of dissociation depends on
the strength of the interaction forces between monomers. These
forces occur at the interface between the B and D chains of the
insulin dimer (see figure at right).

select all
        wireframe off
        ribbon off
        backbone 350
        color chain
        select *.ca or *.cb
        wireframe 100
        select polar and sidechain
        wireframe 100
        color green
        select hydrophobic and sidechain
        wireframe 100
        color blue
        select charged and sidechain
        wireframe 100
        color red
        restrict :b or :d

Now try to see what is holding B and D chains together.
(19)Compare these two images.

P28-K29 in the native is now K28-P29 in the mutant

The in vivo activity of subcutaneously injected insulin is directly related to how fast aggregate preparations dissociate into monomers. The main effect of the mutant has been to weaken the interaction between B and D chains, but to still allow aggregation (dimer and hexamer formation) in the presence of Zn2+ and Cl-. Note that the proline side chains are oriented toward the BD interface in the native structure (left image) but not in the mutant structure (right image). The mutant eliminates an important hydrophobic contact, which weakens the BD interface. As a result, hexamers and dimers of mutant insulin show an increased rate of dissociation into monomers following subcutaneous injection. This leads to a much faster in vivo response and, thus, a so-called rapid-acting insulin preparation. The mutant form is marketed under the name Humulin or Humalog (Eli Lilly).

Best Practices and Tips

Optimizing performance for large molecular structures and ensuring accurate representation are critical in molecular visualization. Here are some best practices and tips for achieving these goals:

Simplify the Structure: For large structures, simplify the representation by hiding non-essential elements such as solvent molecules or side chains in proteins that are not involved in the interaction of interest. This can significantly improve performance.
Use Level of Detail (LOD): Implement LOD techniques to display simplified versions of complex structures when zoomed out and more detailed representations when zoomed in. This can help maintain performance without sacrificing detail.
Utilize Hardware Acceleration: Use hardware-accelerated graphics if available, as they can greatly improve rendering speed and performance, especially for large structures.
Optimize Rendering Settings: Adjust rendering settings such as atom size, bond thickness, and lighting effects to find a balance between visual quality and performance.
Limit the Number of Frames in Animations: For animated structures, limit the number of frames and the complexity of movements to ensure smooth playback and prevent performance issues.
Use Culling and Clipping: Implement techniques such as back-face culling and clipping to remove unnecessary geometry from the view, improving performance without compromising accuracy.
Use a Multithreaded Approach: Utilize multithreading to distribute the rendering workload across multiple CPU cores, improving performance for complex structures.
Avoid Overlapping Structures: Ensure that molecular structures are not overlapping, as this can lead to visual artifacts and inaccuracies in representation.
Regularly Update Software: Keep your molecular visualization software up to date to benefit from performance improvements and bug fixes.
Consider Server-Side Rendering: For extremely large structures, consider using server-side rendering solutions that can handle the processing and rendering on powerful servers, delivering the result to the client for display.

By following these best practices, you can optimize the performance of molecular visualization for large structures while ensuring accurate representation.

Future Directions and Conclusion

Future advancements in molecular visualization tools are likely to focus on several key areas, including:

Virtual Reality (VR) and Augmented Reality (AR): Integration of VR and AR technologies to provide more immersive and interactive experiences for users, allowing them to explore molecular structures in a more intuitive manner.
Artificial Intelligence (AI) and Machine Learning (ML): Utilization of AI and ML algorithms to automate and enhance various aspects of molecular visualization, such as structure prediction, docking, and analysis.
Cloud-Based Visualization: Development of cloud-based visualization platforms that can handle large-scale molecular datasets and provide collaborative tools for researchers worldwide.
Enhanced Interactivity: Continued improvement in the interactivity of visualization tools, allowing users to manipulate and analyze molecular structures in real-time with greater ease and flexibility.
Integration with Big Data: Integration of molecular visualization tools with big data analytics platforms to facilitate the analysis of complex biological data and enable data-driven discoveries.
Improved Accuracy and Realism: Advancements in rendering techniques and algorithms to achieve higher levels of accuracy and realism in the visualization of molecular structures.

In conclusion, molecular visualization is a powerful tool that plays a crucial role in understanding the structure and function of biological molecules. By following best practices and leveraging advancements in technology, researchers can optimize the performance of molecular visualization tools for large structures while ensuring accurate representation. The key takeaways from this tutorial include:

The importance of molecular visualization in understanding complex biological processes.
Best practices for optimizing performance and ensuring accurate representation of molecular structures.
The significance of tools like RasMol and potential future advancements in molecular visualization technology.

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