Bioc585 2008 Exercise 1 – Coot Homework due Tuesday, April 1 We will spend the first two computer sessions on exercise 1. The main goal of this exercise is for you to become familiar with the molecular graphics display program Coot. To accomplish this, you will explore the protein human thioredoxin after mutation and modification. Thioredoxin is a small, redox active protein that undergoes reversible disulfide bond formation, involving Cys 32 and Cys 35, in its active center. There are 5 cysteines in the protein, out of a total of 105 amino acids. In the present experiment, Cys 73 was mutated to serine, the protein was purified and crystallized, and the oxidant H2O2 was added. The resulting crystal was flash frozen and diffraction data were measured in-house to 1.35 Å resolution. The previously analyzed structure of the wild-type reduced protein, PDB entry 1ERT, was used as a starting model and refined against the new data for a few cycles to give a preliminary model of the modified protein. We will run all exercises in the Linux computing environment. To begin, if necessary, reboot your computer and select the Linux multiuser operating system. If you are unfamiliar with Unix/Linux, do not fear: there are many tutorials available and I will provide help. To begin, you may want to read the tutorial linked here: http://biochem.arizona.edu/xray/. All files for this exercise are in directory ../shared/585xray/ex1. The files we will use are 1ERT.pdb (starting model), 1ERT_refmac1.pdb (refined model) and ScalAveraged_refmac1.mtz (binary file with diffraction data and preliminary phases). Please copy these to your area as described below – do not work in the shared directory or delete these files! 1. Login to your Linux account (password is password for now). Type the following commands to set up your directory and begin coot. The Coot reference manual can be found on the Macromolecular Crystallography web page: http://biochem.arizona.edu/xray/. mkdir ex1 (creates a new directory) cp ../shared/585xray/ex1/* ex1 (copies files into directory ex1) cd ex1 (changes your working directory to ex1) ls (lists the files in this directory) coot (starts the 2. In coot, use the open coordinates option (File menu) to read in the coordinates. Learn to maneuver through use of the mouse and keyboard commands, highlighted under Hints (Help menu). I like the Flat option (HID / virtual trackball menu). 3. Explore the options under the Draw and Display Manager menus. 4. Center on residue Cys 32 using the Go to atom option (Draw menu). Select the Show residue environment option (Measures / Environment Distances menu). Note the distance between sulfurs in Cys 32 and Cys 35.5. Use the Cell & Symmetry option (Draw menu) to display more of the crystal. Click the Show Symmetry, Calpha and Show Unit Cell options. Explore the options to become familiar with the crystal. Note that some atoms of an individual molecule can be outside of the unit cell box. These atoms are also in the unit cell box, but connected to a different molecule (that is, to obtain a fully connected molecule, one must usually take atoms from more than one unit cell). 6. In Cell & Symmetry, display all atoms. Make sure the Show residue environment option is on. Center on residues 72, 73 and 74, noting close contacts with the neighboring molecule. 7. Calculate electron density maps using the Auto open MTZ option (File menu) and selecting the file ScalAveraged_refmac1.mtz. Three maps will appear, a blue map showing the best overall electron density cage (called a 2Fo-Fc map), a green map showing errors where something is missing in the model (positive region in an Fo-Fc map), and a red map showing where something is misplaced in the model (negative region in an Fo-Fc map). The program will run faster if you turn off the Dragged Map and Smooth Recentering options under Display Manager menu. 8. Center on Cys 73 and examine the maps. Note whether the mutation appears to have been made. 9. Center on Lys 81. Note any changes that might be suggested for the side chain nitrogen. 10. Turn off the maps (Display Manager menu). Read in 1ERT.pdb. Superimpose 1ERT onto 1ERT_refmac1 using SSM superpose (Calculate menu). Make 1ERT_refmac1 the reference structure and 1ERT the moving structure. Note any differences between the structures, particularly in the active site.Homework 1 (10 points) Please answer the following questions. Please justify all conclusions. 1. Do the active site cysteines appear oxidized or reduced? 2. Does thioredoxin appear to be monomeric or dimeric in the crystal? 3. How many thioredoxin molecules are in the unit cell? In the asymmetric unit? (Hint: the space group is C2; there is one chain in the PDB file.) 4. Can you conclude that the Cys 73 mutation was successful? 5. Describe how you would change Lys 81 in response to the electron density maps. Bonus question (0 points, but you will achieve enlightenment): Use the Edit Chi Angles option (under Calculate and Model/Fit/Refine menus) to change the Lys 81 position as suggested in question 5. Obtaining Coot The Coot software is open source and can be loaded onto most computers. The URL is: