1 Bioc585 2008 Exercise 3 – Calculating Electron Density Maps using CCP4i and Coot Homework due Tuesday, April 15 It is now possible to use menu-driven crystallography programs on a PC to calculate structure factors or electron density maps that can be read by modeling programs. The package we will use is called Collaborative Computational Project Number 4 (CCP4) and is available with a Graphical User Interface (GUI) called CCP4i. We will make considerable use of this package. First goal: To convert intensities to structure factor amplitudes, perform an initial refinement with a starting model, and view 2Fo-Fc and Fo-Fc electron density maps. Second goal: Calculate “omit” maps for interpreting questionable model features. Overview 1. Obtain coordinate and diffraction data 2. Determine the format and content of the diffraction data 3. Start CCP4i and convert the diffraction data to an MTZ file 4. Calculate structure factors (amplitude and phase) by Fourier Transformation of the coordinates 5. Calculate a 2Fo-Fc electron density map by Fourier Transformation in the opposite direction, and view the map, using Coot. 6. Modify the PDB file and calculate and interpret Fo-Fc and 2Fo-Fc electron density maps. 1. Obtain coordinate and diffraction data. Coordinate and diffraction data files can come from a variety of sources, but in most cases will be provided by a local crystallographer or found at the PDB. For this exercise, we will use PDB file 1ERT (reduced human thioredoxin) and a local data set called trx_h2o2.ref. Both are text files that can be opened in gedit (Accessories > text editor), and both are in the shared directory. Create a sub-directory (folder) called trxh2o2 in your disk area and copy these files into this folder. 2. Determine the format and content of the diffraction data. Diffraction data files, unfortunately, are not yet standardized. Thus, it is important to understand what is in the file before proceeding. In the present case, the data are the output from a program called d*trek, which is a format recognized by CCP4i, and already scaled and merged. In many cases, particularly when obtaining data from the PDB, this will not be the case and more care must be taken with importing the data – this will be covered in a later exercise. Print the first few lines of the file to the screen by typing “more trx_h2o2.ref” and “q” to quit. The first few lines of the file should look like this: 3 2 0 5 CRYSTAL_DESCRIPTION=unknown; CRYSTAL_MOSAICITY=0.4115 0.0000 0.0000; CRYSTAL_ORIENT_ANGLES=93.7259 -9.5125 82.9509; CRYSTAL_SPACEGROUP=5; CRYSTAL_UNIT_CELL=66.1665 26.3751 51.0998 90.0000 95.4684 90.0000; nH nK nL fIntensity fSigmaI 0 2 0 170058.0 1943.50 0 2 1 146273.9 2886.86 0 2 2 35688.5 527.1202 The first 6 lines provide information about the crystal, including unit cell constants and space group, and the next 5 lines describe the values found in each data line, in this case HKL, I and σI, where HKL are integers and I and σI real numbers. The next 3 lines are the first of several thousand data lines. For example, the first line lists I020 = 170058.0 (1943.5). 3. Start CCP4i and convert the diffraction data to an MTZ file. Information on CCP4i is in Appendix 2. To convert your data: A. Start CCP4i by clicking on the icon, go to menu Directories&ProjectDir (on the right): B. Add a new project with the directory name where your files reside, e.g.: *** Note: if you select an item and then right click, information on the item is displayed. ***3 C. Click the Data Reduction menu and select Import Merged Data. In this menu: (i) Choose d*trek (ii) Unclick Use anomalous data. (iii) Choose Run Truncate. (iv) Select the appropriate file name (using Browse). (v) *** Be sure to turn off data harvesting option *** (vi) Note that the output file name has already been assigned (can be changed), and that the cell parameters were read from the input file. (vii) Add the wavelength (copper Kα , 1.5418 Å) and the number of residues (105). Include crystal and dataset names (these are not important for our purposes, but the program expects values).4 (viii) Run the program. Close this window and return to the main window. The output can be viewed by selecting the job and clicking on the View Files From Job button. 4. Calculate structure factors (amplitude and phase) by Fourier Transformation of the coordinates. There are many ways to accomplish this. Perhaps the most useful is to use the refinement program Refmac for this, since we will use this program for other purposes and the program output feeds directly into Coot. We will cover refinement in more detail later, but for now, simply note that the refinement algorithm shifts atom positions in the model such that the predicted and observed diffraction patterns better agree. Set the menu up like below. Note the number of cycles.5 5. Calculate a 2Fo-Fc electron density map by Fourier Transformation in the opposite direction, and view the map, using Coot. Read your new MTZ file (trx_H2O2_refmac1.mtz) into Coot, as before. 6. Modify the PDB file and calculate and interpret Fo-Fc and 2Fo-Fc electron density maps. We will alter the structure to address the issues raised in the last session, and interpret the results. A. Fix the position for Lys 81 using the Edit Chi Angles routine in the Model/Fit/Refine menu (click on an atom in the residue). B. Mutate residue 73 to serine using the Simple Mutate command (Model/Fit/Refine menu; click on an atom in the residue) and place the side chain in the best position using Edit Chi Angles. C. Alter the occupancy of Cys 32 so that the side chain atoms have zero occupancy (zero electrons). Do this by selecting Residue Info (Measures menu) and clicking on an atom in the residue. Edit the occupancies in the dialog box to read 0. D. Save the new model in your directory (Save Coordinates routine, File menu). Refine the new model using Refmac. Read the new model and new MTZ file into Coot and answer the homework questions. Homework 3 (10 points) (1) Describe the electron density for Lys 81 before and after model building and refinement. Why are the maps different after model building? Include a picture of the final 2Fo-Fc