Basic PyMOL Tutorial PyMOL is a great freeware program used to view 3D crystal structures created by Delano Scientific, and could be downloaded for free previously. If not, you can purchase it at https://www.pymol.org/ ​ The freely available version is 0.99, although the newest version is 1.0. Students and Professors can download the new version only after you have registered and the company approves you. For everything we do, I have not found that version 0.99 is in anyway obsolete. Step 1 PyMOL takes .PDB files which can be found at http://www.pdb.org by ​ ​ searching for the molecule of your choice. In the example shown below, I have located the C8γ-Indel structure and I would like to download the .PDB to view and manipulate in PyMOL. The .PDB file can be downloaded by navigating to the left pane under Display Files, then right clicking on the the PDB File, and then Save Link As…as shown circled in red. After you have saved the file in your ​ ​ desired location, you can now open PyMol and view the structure. 1 Step 2 Open the previously installed PyMOL application, navigate to the file menu, and then open the newly downloaded structure (in this case, C8γ-Indel with 2qos accession code) as shown below. The molecule will open in the Line form, with all atoms in the backbone and ​ ​ sidechains shown by carbon in green, nitrogen in blue, and oxygen in red. Waters and any bound ligands will also be shown at this point but may be difficult to decipher from the protein in Line view. Since I find the Line view rather useless, we will change to the more common Cartoon view to look at the structure. This is achieved by navigating to the right menu show below where each letter stands for A=Actions, S=Show, H=Hide, L=Label, and C=Color. 2 We must first Hide all lines, including the proteins, waters, and in this case no ​ ​ ligands. This is done by clicking on the H button next to the all tab , and then ​ ​ clicking on Everything. This will give you an entirely blank screen with no ​ ​ structure shown at all. To Show the structure up in cartoon mode, click on the S ​ ​ ​ button next to the all tab, and then click cartoon. This will give you a structure as shown below: NOTE: If you would like to switch to a Line, Stick or Ribbon view, you must first Hide the Cartoon view, or the new view will be overlayed on the current cartoon. Step 3 To enjoy all of the features of PyMOL you will need a 3-button mouse. The buttons functions as follows: Left button: Rotates the protein around a center axis. ​ Center scroll wheel: When held down will move the protein around the screen as ​ an object. 3 Right button: When held, will zoom in on the protein by pulling the mouse ​ towards you (zoom in), and pushing away (zoom out). I chose to use the C8γ-Indel structure because it not only incorporates a protein, but a peptide too. This means that there are really 2 objects in the structure, but they will be called 2 different Chains from here forward. In the case of this ​ ​ structure there is an A and a C chain which stands for the C8α Indel peptide, and ​ ​ C8γ protein respectively. The chains are named after the greek letters α, β, γ, ​ ​ which stand for a,b,c) Now that you know how to rotate, zoom and move the molecule, changing color is next. There are two ways to change the color; by using the right tool bar under C=Color, or using the command line located in the window labeled PyMOL ​ Td/Tk GUI as circled in red below. If you would like to color both the A and C ​ chains orange, you would type “color orange” in the command line: 4 Notice that with this “color orange” command, you will color both C8γ and the C8α Indel which is circled in green. To alleviate this problem, there is a special ​ ​ command for coloring only one specific chain. I will use the following command to change the C8α Indel peptide to green, while keeping C8γ orange: “color green, chain A” which produces the following screen: Likewise, if you would like to chain the color of C8γ to red, you would use the command “color red, chain C”. 5 Changing the color of specific residues: Command are also helpful to color ​ specific residues in a chain different than the main cartoon color. The following command would be used to color residues 55-110 purple in C8g: “color purple, chain C and resi 55-110” To give the following: Now that you know how to manipulate the color of individual chains, let’s take a look viewing side chains of individual residues. 6 Viewing Side Chains: There are several ways to identify an individual residue ​ that you would like to look at. The first way is to directly left click on the molecule, and the residue that is highlighted will be identified in the PyMOL Td/Tk GUI window as shown here: You can see I click on Glu 45 as shown in the text window. At this time, you can show this side chain by navigating to the right tool bar, where you will now see a tab labeled (sele), which stands for the residue you have just selected. Now under the (sele) tab, click on the S button for show, and then ​ ​ the side chain tab, selecting to show the sticks representation. The side chain will ​ ​ ​ ​ be the same orange color as the cartoon back bone. You can change the color by navigating to the (sele) tab once again, and clicking the on the C button for color. You can choose to color the sidechain a solid color, or you can color the side chain by element. I like to color side chains by atom because nitrogen is blue, oxygen is red, and you can state what color you want carbon to be. Shown below, I have made carbon yellow by coloring by element: 7 While this works very well for coloring the side chain, as you can see, the backbone carbon was also colored yellow, taking away from the orange flow the backbone. This can be fixed by only changing that one carbon back to orange. To achieve this, hold down the Control button on your keyboard while right-clicking on the carbon. You will see the following white sphere around the carbon if done correctly, and the presence of a new toolbar to the right: To chain the backbone back to orange, navigate to the new (pk1) tab and clik on the C button for color. Change to orange, and you now have an orange backbone 8 with a nice yellow side chain. To remove the white sphere from the carbon, you have to hold control and right click away from the protein molecule. Changing the background color: You can change the background to white to ​ make figures by navigating to the top toolbar under Display, then Background, ​ ​ ​ ​ and choose white. I will work with a white background from here forward. Making the cartoon backbone transparent: There are times that you would like to ​ highlight the side chains and not have the cartoon backbone take away from the figure. A nice way to do this is by making the cartoon transparent. This can be done by navigating to the top tool bar under Setting, then Transparency, then Cartoon, and choosing your desired transparency level. I like to use 60% as shown below. You can see that the cartoon look changes, indicating the transparency setting has taken effect. This grainy look with 60% transparency will not be present in the final figure I will be showing you how to create later in this tutorial. 9 How to create high-resolution figures: To create high-resolution figures worthy ​ of publication, you must perform what is known as a ray function. This can be done by creating a figure you are ready to Ray, and the typing the following command into the command window: “ray 1200,1200” This will create a figure that is 1200x1200 pixels, which is very high-resolution. This function may take a few minutes as shown by a progress bar, but will produce a screen that looks like the following. You can see the cartoon transparency compared to the sidechain that has very bold colors. 10 At this point, you must save the picture by going to the File, Save image as.. tab. ​ ​ ​ ​ Images will be saved as .PNG image files. You should also save the work you have done with this molecule by going to the File, Save session tab. This file will have a .PSE extension, and can be opened ​ ​ ​ only by PyMOL so you can pick up where you left off while manipulating the shown C8γ-Indel structure. More advanced options will not be covered in this tutorial. A detailed tutorial directly from the software author can be found here: http://pymol.sourceforge.net/tutorial/index.html Another VERY helpful tutorial that covers molecular surface, bond distance and making movies can be found here: http://137.189.50.96/kbwong/teaching/pymol/pymol_tutorial.html 11 .