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sure Be to modify as per modified LG Tutorial! Welcome to the PyMOL Tutorial! Written by Jae Woo Choi, Wayne Gui, Trevor Halle, Mohamed Khattab, Priya Malhotra, Dmitriy Migdalovich, Cina Sasannejad, Seung Youn Shin, Rob Tirrell, and Jerry Feigenson Introduction: This molecular graphics part of the biochemistry course will give you the power to examine the structure of many thousands of molecules-- large polymers such as HIV protease, DNA, and RNA, and small molecules such as estradiol, iboprofen, and ATP. PyMOL is a software program used to display and examine 3-D representations of biomolecules. Why the name PyMOL? It was written in the Python programming language. Though not required for this class, you can learn more about PyMOL at the website, Table of Contents: Basic Features: 1. Set-Up (Inserting PyMOL CD, Unzipping Files) 2. Introduction to Menus (Interface) 3. Menu Buttons 4. Reading Directions in PyMOL, Help, and Wiki 5. Using the Mouse (Windows, Mac) 6. Building Polypeptides Tutorial Exercises: 1. Myoglobin 2. Ebola Virus 3. Antibody Fragment Binding to a piece of HIV-Protease Tutorial: 1. Insert the PyMOL CD. Drag the 1&2 PyMOL folder to your desktop (or Copy and Paste it). To open: Windows: Unzip the file, then double click on SETUP.exe, then to open the application, click on "PyMOL + Tcl-Tk GUI" wherever it has been installed. Note that older versions of WinZip might not work easily. If the program does not seem to start, restarting your computer may fix the problem. Macs: Double click on MacPyMOL.tar.gz. The MacPyMOL application icon will be created on your desktop. Drag this icon to your applications folder. 2. In order to get a simple screen to start this tutorial: From the menu at the very top of the screen, choose Build > Residue > Alanine 1 Menus There are two menus: 1) The ones at the very top of your screen are the main program menus. These can be used to open and close structure files, and perform general commands. 2) The other important menus are located at the right of the PyMOL program in the long rectangle. 3. We will now examine how to use the menus on the right: There are six buttons in a row; one long rectangular button followed by five small square buttons labeled The first one is a label naming what you want to manipulate. For example, when it says "all", that means you will manipulate every part of the molecule that is open. When it says "<sele>", you will manipulate only what is selected (more about 'selecting' below). The next five buttons allow you to manipulate the protein or parts of the protein depending on which menu button you choose: A Actions. With this menu, you can perform general actions such as adding or removing hydrogens, or removing water molecules. S Show. With this menu, you can show the protein in a particular display such as spheres, lines, sticks, ribbon, or 'cartoon'. With Alanine now displayed in "lines", select the 10th choice, spheres. H Hide. This menu allows you to hide the part of the protein that is in a particular display of spheres, lines, sticks, ribbon, or cartoon. If you want to display something in spheres and it is already in cartoons, you must hide the cartoons. Otherwise the protein will be in both spheres and cartoons, which will be confusing! 2 L Label: This menu allows you to label the protein in a certain way in case you want to note what certain residues or atoms are. For example, you can label a residue as Ala or label an atom as C. C Color. With this menu, you can color whatever is selected just about any color you can imagine. There is even Pea Green! NOTE: An important color in this menu is under "by element" It is the 2nd CHNOS down. Each letter represents an element and is colored the way that element will be displayed. The 2nd one down colors the carbon green, nitrogen light blue, oxygen red, sulfur orange and hydrogen gray. Another important color display is "by chain" which will color each peptide chain of the protein a different color in order to distinguish the different chains. NOTE: 4. READING THE DIRECTIONS IN THESE PYMOL EXERCISES Command Line There are two command lines: 1) One is in the grey window at the top 2) The other is in the black PDB view window at the bottom of the screen We suggest always using the top command line because you can see if an error has occurred in your command or if your command went through okay. TAKING SCREENSHOTS OF IMAGES IN PYMOL: 3 You may find it useful to take screenshots of images in PyMOL for your future reference. Windows: For best results, maximize the image window and then press the "Prt Sc" (Print Screen) button in the top right corner of the keyboard. Then return to the word document and paste ("Ctrl + v") the image. Make sure to save these changes to the document. Macs: For best results, maximize the window and then press "CommandControlShift3" to save the image to your clipboard. Return to the word document and press "Command + v" to add the image. Make sure to save these changes to the document. *IMPORTANT* In what follows below, whatever is after "PyMOL >" is a command and must be typed or pasted into the command line in the program. Note that only the part after "PyMOL >" should be typed into the command line. Any other instruction means to use the drop down menus. Directions will be shown in the font Courier New. Help: Help for PyMOL is on the PyMOL Wiki website at Also, if you put "help" (all lower case letters) into the command line in PyMOL and press enter, the output will list important PyMOL commands. To get help about a specific PyMOL command, put "help command Name" into the command line in PyMOL and press enter. IMPORTANT In order to open new PDB files, you can just double click the new PDB files or "reinitialize" the old one and then open the new PDB file in the same window. If you do not reinitialize, you will have several PyMOL images open at the same time. In order to reinitialize, go to File > Reinitialize 5. DIRECTIONS FOR USING THE MOUSE: a) Three-button mouse (default setting): Left Button: Rotates Molecule Right Button: Zooms in and out Middle Button: Moves the molecule around the plane of the screen Control Shift and Middle button: Set the origin of rotation Shift and Right Button: Clipping (chops off a plane of the molecule) Single Click Middle Button: Sets Center b) For users with a two-button mouse: Go to Mouse > 2 Button Viewing Mode Left Button: Rotates Molecule Right Button: Zooms in and out Ctrl Left Button: Moves the molecule around the plane of the screen Shift Right Button: Clipping 4 Double Click Right Button: Sets Center The three button mouse is default, so if you need to use a two button mouse, every time you open the program, you must set the mouse setting to "2 Button Viewing mode." Do this after a file has been opened. NOTE: c) For users with a one-button mouse (Mac users): Mouse > 1 Button Viewing Mode Button: Rotates Molecule Ctrl: Zooms in and out Ctrl shift: Clipping Option/Alt: Moves the molecule around the plane of the screen NOTE: The threebutton mouse is default, so if you need to use a one button mouse, every time you open the program, you must set the mouse setting to "1 Button Viewing mode." Do this after a file has been opened. 6. BUILDING POLYPEPTIDES: Before building polypeptides, you should get better acquainted with the different displays that are available on PyMOL. To do this, go to Menu > Wizard > Demo > Representations. Now, go to Build > Residue > Alanine. Then repeat Build > Residue > Glycine to add Glycine, and repeat to add a Histidine residue. Then apply the following: <all> > S > sticks <all> > C > by element > CHNOS (2nd one down) Be able to recognize each of the amino acids and the two peptide bonds joining them together. Notice between which atoms the peptide bonds are formed. If you have trouble identifying where the peptide bonds occur, you can have PyMOL label atoms according to the residue containing them. To do this, select: 5 <all> > L > residue name To turn labels off, select: <all> > L > clear Now, put the peptide you have built in spheres mode: <all> > S > spheres In the top right corner of the gray window, click on the "Rock" button. (You might need to click again on "Rock" in order to turn it off). Now click on the "Ray" button. The Rock button will rotate the image from left to right while the Ray button will enhance the image's resolution. See how these features convey the 3-dimensionality of the structure. However, also notice that in spheres mode, recognizing the peptide bonds and even the individual atoms is far more difficult than in the less realistic sticks mode! Note: It might prove useful to create images that you can to refer later on if you need to review your PyMOL exercise. For best results, maximize the size of your PyMOL image, take a screenshot of your molecule, and "paste" the image into the PyMOL Word document. Next, save the altered image so you can return to it in the future. Congratulations! You have learned the basic mechanics of PyMOL. Now we will go through a few examples in order to learn important commands that you will use throughout the semester in the PyMOL assignments. The examples are Myoglobin, Ebola Virus, and HIV protease. Refer to the directions above when loading PDBs, using mouse controls, and searching through PyMOL help. Exercises: (1) Myoglobin (Mb) PDB stands for Protein Data Bank. Many molecules, both large and small, have the PDB format. Mostly, you will use the PDB file format. A PSE file is a session file that has been saved in PyMOL. It is like a PDB file, but has been altered to display the protein in a particular way. For Macs, to open a PDB/PSE file, first reinitialize the PyMOL program, then open the file via the menu at the top of the screen, File > Open. (Note: To open a second PyMOL window using Macs, right click on the PDB/PSE file you want to open, then scroll to "Open With" and choose MacPyMOL. Or, make a copy of the MacPyMOL application, and open the second file in the new window). In Windows, you can open the PDB/PSE file by double clicking on it, whereas in Macs you must go to File > Open in order to open the file. (Note: In order to open a second PyMOL window using Windows, just double click on the PDB/PSE file that you want to open). Loading Myoglobin: 6 File > Reinitialize File > Open and choose 1A6M (This file is in the 1&2 PyMOL Folder > 1-Tutorial Folder). Commands: Examining Myoglobin: To display a selected part of the molecule in a certain way, type "select" into the command line. Whatever you select will appear as "<sele>" in the right menus, and tiny pink squares appear. In order to manipulate this selected part of the molecule, go to the menus beside "<sele>". Go to the C menu to color this part of the molecule, then go to S in order to show the molecule in a particular display of your choice. In order to hide the part that you just selected, go to H and click on the display you want to hide. For example, let us work with the heme group of the Mb. First, hide the waters: Go to the <all> tab and click on A and remove waters Then go to the command line and type in: select organic To display the heme group in spheres, go to the <sele> tab, then click on the S button and click on spheres. To hide the rest of the molecule, go to the <sele> tab, click on the H button, and click on unselected. To color the heme group by element: Click on the C button near the <sele> tab, then click on by element, then click on CHNOS (2nd one down). Now you have isolated just the heme group of myoglobin! Summary of Automated Commands: <all> > remove waters PyMOL > select organic (Remember that anything after "PyMOL >" is a command and must be typed or pasted into the command line in the program.) <sele> > S > spheres <sele> > H > unselected <sele> > C > by element > CHNOS (2nd one down) This should show the heme group of Mb in Spheres and CPK colors 7 Residue: In order to select a residue, you will select the residue number, then you will show the atoms in a particular display, and lastly you will color them a particular color. Currently, we have just the Mb heme ligand in the PyMOL view. Now we will show a particular residue. Type or paste the following command into the command line and do the rest of the menu directions: PyMOL > select resi 87 Nothing will happen on the Display screen when you do this, but a new tab will appear on the right called <sele>. <sele> > S > sticks <sele> > C > blues > blue This will show residue 87 in sticks and blue. If you click on the residue, you can find out information about it in the top gray window. You clicked /1A6M///LYS`87/CG This shows that you clicked on residue 87, which is a lysine. NOTE: Anything selected will appear as <sele>. Before, you selected organic. Now you selected a residue. A new <sele> will not appear, but instead will replace what was selected before. Therefore, there will still be one <sele> on the right menu, but it will be the residue instead of organic. Distance: Now we will measure the distance between an atom on the Lysine and an atom on the heme group. In order to get the measurement, go to the PyMOL menus at the top of the window, and click on Wizard > Measurement. Then click on first one, then the other of the two atoms you want to measure the distance between. In order to finish taking the measurements, go to the right, and underneath Measurement, there is a gray button that says Done. Click on this. The display should look something like this (Note that your exact measurement will depend on which atoms you chose): Transparency: To show the rest of the protein again: <measure01> > A > delete <all> > S > main chain > sticks 8 This got rid of the measurement label and showed the rest of the protein in sticks, not including the heme group. In order to make one part of the Mb stand out, you can make the rest of the molecule transparent. Let us make the heme stand out. In order to do this, type into the command line: <PyMOL> > set stick_transparency, 0.6 NOTE: Choose a number between 0 and 1, where 1 is transparent and 0 is opaque. 3-D Visualization: Reinitialize and load Myoglobin again: File > Reinitialize File > Open and choose 1A6M. Remove waters then put Myoglobin in spheres view: <all> > A > remove waters <1A6M> > S > spheres Next, go to Display > Stereo On and Display > Stereo > Cross-Eye Stereo. Cross your eyes and focus on the area between the two Myoglobins. This is similar to 3-D puzzles where objects appear after staring at a picture cross-eyed. Do you see Myoglobin in 3-D? This can be difficult, so do not worry if you cannot see it. (2) Ebola Virus Reinitialize and Load the Ebola virus PDB: 1ES6 Use the following directions to see which parts of the Ebola Virus are more hydrophilic and which parts more hydrophobic. <all> > A > remove waters PyMOL > select hydrophobes,(resn ala+val+ile+leu+phe+met) <hydrophobes> > S > spheres <hydrophobes> > C > yellows > yellow PyMOL > select hydrophilics, (resn arg+lys+his+glu+asp+asn+gln+thr+ser+cys) <hydrophilics> > S > spheres <hydrophilics> > C > blues > blue As you can see, there are many hydrophobic residues on the surface of the Ebola Virus, which provide affinity for the cell membrane of the host. Notice the residues that do not have polarity. Which are the residues sticking out of the molecule? Try saving a copy of this image in the word document. Refer to the section "Taking Screenshots of Images in PyMOL". Clipping: 9 Since a biomolecule is a complicated 3-D structure, it can be useful to examine different slices of the molecule in order to gain a better understanding of its structure and function. The clip function is used to trim away layers of the molecule in order to view what is beneath. To clip, first go to Display > Two Sided Lighting. For a 2-button mouse, move the mouse while holding the shift key and the right button of the mouse. A three button mouse allows you to clip by just rotating the mouse wheel. Use clipping to see if the ebola virus is more hydrophilic or hydrophobic on the inside of the protein (3) Antibody fragment binding to a piece of HIV-protease Reinitialize and Load the Antibody & HIV-Protease PDB: 1JP5 There are two antibody HIV-Protease complexes in this PDB file that consist of four polypeptide chains. Chain a and chain b are the antibody fragments, and chain c and chain d are 9 amino acid fragments of HIV-Protease. In order to select one of these complexes, follow these directions: <all> > A > remove waters <all> > S > lines <all> > H > sticks PyMOL > select chain a <sele> > C > reds > red PyMOL > select chain c <sele> > S > spheres <sele> > C > by element > CHNOS (2nd one down) This should show the antibody tightly fitting to the piece of HIV-Protease. As a review exercise, try transparency again and make the HIV-protease stand out. PyMOL > set stick_transparency, 0.75 10 In order to modify any previous command, it is NOT necessary to again type all the characters! First, retrieve the previous command simply by pressing the "up arrow" on your keyboard. Then modify the part of the Command Line. Here, use the up arrow to again see "set stick_transparency, 0.75". Now change the value to 0.3 and hit Enter. YOU NOW HAVE A BASIC UNDERSTANDING OF HOW TO USE THIS POWERFUL MOLECULAR GRAPHICS SOFTWARE! 11 ... View Full Document

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