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Chem 240 Fall 2011 1 The Basics of UCSF Chimera This is a very basic introduction to UCSF Chimera, a powerful, free software package for visualizing and analyzing macromolecules. If you want to explore Chimera further after running through this tutorial, check out the Chimera user’s guide, at: www.cgl.ucsf.edu/chimera/docs/UsersGuide/index.html To begin, download and install the latest production release of UCSF Chimera (currently 1.5.2): www.cgl.ucsf.edu/chimera/download.html ___________________________________________________________________________ Part One: Loading structures and basic viewing Open Chimera. Chimera can read several file formats, including PDB, or “Protein Data Bank”. The PDB is a collection of structural data on proteins (and other biomolecules) and can be found online at rcsb.org. It’s awesome. Any one of the 63,000+ structures on the pdb can be downloaded directly into Chimera if you know the pdb code. For this exercise we’ll use a structure of insulin, pdb code 3i3z. Here’s how to load it in Chimera: From within Chimera….. Go to File/fetch by ID Type in 3i3z and click “Fetch” After a moment, a structure will appear on the screen. This is the peptide hormone insulin. Take a moment to familiarize yourself with the controls. To rotate the molecule, left‐click and drag. To zoom in and out, right click and drag OR use the scroll‐wheel To translate the molecule in the plane of the screen, using a 3button mouse, middle‐click and drag On a Mac, or on a PC without a middle‐mouse button, we’ll have to change the default controls: Favorites/preferences Category: Mouse For the translate x,y column, click the Ctrl2 button, as shown below: Chem 240 Fall 2011 2 Click Here Now you can translate in the x,y plane by holding “CTRL” on the keyboard and right click‐dragging with the mouse. There are a lot of options available from the menus on the top of the window. Still more are available through the command line, which you should enable: Favorites/Command line The bottom of the window should expand to reveal the word “Command:” with an empty space next to it. The command interface can be very powerful, enabling you to even script your own molecular movies. But right now, I only want you to remember one command: focus When you type focus in the command line and hit enter, it brings the molecule back into the center of the screen. Very useful if you get lost while rotating and translating. Answer the following questions and email them to your instructor. Chem 240 Fall 2011 3 Part 2: Visualizing Insulin You’re looking at the protein in ribbon form, in which a ribbon traces out the backbone of the protein. Notice that if you hold your mouse over any part of the structure and wait for a second, a window pops up with information about what is directly under your pointer. Hold it over a part of the protein and you’ll see the 3‐letter code for the amino acid at that spot along with its sequence # and chain ID. This structure has two chains, so depending on where you hover your mouse you might see info on an amino acid from chain A or chain B. Using this technique, answer these questions: (1) What is the first amino acid of Chain A?_________________ What is the last amino acid of Chain A?_________________ What is the first amino acid of Chain B?_________________ What is the last amino acid of Chain B?_________________ Now let’s turn on the side chains: Actions/atomsbonds/sidechainbase/show The backbone is still treated as a ribbon, but now the sidechains should be projecting outward. The command line can be used to select individual residues, either by sequence number, or amino acid type, etc. Type this in the command line: sel :29.B Press enter. An amino acid should become outlined in green. What residue was selected? Give the full name, 3‐ letter abbreviation and one‐letter abbreviation: (2) ____________________________________________ Control click outside of the molecule to unselect. Chem 240 Fall 2011 4 The default display for atoms in Chimera is “stick”, which is great for seeing the geometry of a molecule, but not so great for getting an idea of how much space an atom occupies. Try out the various other display options for atoms and bonds: Actions/atoms bonds (and then choose either wire, stick, ball and stick, or sphere) Note: as you do this, remember that the backbone atoms are still hidden…the backbone will stay as a ribbon as the sidechains change their appearance. As you’ll see, sphere displays atoms based on their Van Der Waals radii, which is a good approximation of an atom’s size. Switch to sphere mode. Comment on the colors of spheres in this depiction: (3) What does red signify?_______________ Blue?_______________ Grey?________________ Yellow?_________________ Comment on the distribution of colors in the structure. Are non‐grey atoms found evenly distributed throughout the structure, or are they mostly on the inside or outside? Explain the sensibility of your observation in terms of water solubility. (4) Are there any hydrophobic patches on the exterior of insulin? If so, give an example of a specific amino acid in this patch, including its sequence number and chain ID. (5) Switch back to stick view. Take a close look at the yellow bonds that connect the two chains of insulin. What types of bonds are these, and how many of them exist in this structure? (6) Chem 240 Fall 2011 5 Lets finally take a look at the shape of the whole protein. To do this we’ll use the surface command, which is like putting shrink‐wrap over the spheres that represent each atom in the molecule. This is probably the best way to get an idea of the overall shape of a protein, including binding pockets and crevices. Actions/surface/show Move the structure around a little. You’re looking at the solvent‐accessible surface area of the protein. In other words, the parts of the protein that water can “touch”. Notice all the blue and red! In this depiction, the surface is colored by atom identity; that is, if an atom is carbon it gets one color, if its oxygen it gets another, etc. We actually have lots of coloring options in Chimera to help us see patterns in a molecule…one useful approach is to color by the hydrophobicity of each amino acid. There’s a shortcut for this one: Presets/interactive 3(hydrophobicity surface) Here orange is hydrophobic, blue is hydrophilic and white is considered in between the extremes. Notice that big hydrophobic patch again. Insulin in its inactive form is a hexamer. The hydrophobic patch helps it to clump together to form these larger, 6‐member complexes. To make the surface slightly transparent so we can see the backbone ribbon inside, Actions/Surface/Transparency/70% (or some other %...play around a bit) Save an image of this scene and email it to your instructor along with the answers to the questions above. To save an image: File/save image/Save as/save The default image options are fine. A final note on saving work: You save your work in Chimera as a “session”. Its not required for this exercise, but here’s how to do it: File/Save Session As… Note that if you want to reopen a Chimera session, you can’t double‐click the session file like you would a word document. You have to first open Chimera, choose File/open from within Chimera and then navigate to the session file (it will have the extension .py).