X-ray Crystallography of Peptides and Peptidomimetic Compounds

Tools Needed for the Crystallography Exercise

For this exercise we will be using two crystallographic programs: Phenix and Coot. Phenix is a crystallography suite that will allow us to analyze diffraction data, generate density maps, and refine and validate our structures. Coot is an interactive model building program that allows us to modify, add, and adjust our model to the electron density map.
Phenix is available for Mac, Pc, and Linux here: Download Phenix
Coot is available for PC here: Download WinCoot
Coot is available for Mac here: Directions for installing on Mac

X-ray Crystallographic Data Set

The X-ray crystallographic data set for this exercise is available here: Crystallography Exercise Data Set
The ZIP file contains an unmerged diffraction data set (data_set_unmerged.ahkl), a merged diffraction data set(data_set_merged.mtz), retraint files for unnatural amino acids (ORT-FINAL.cif, PHI.cif, and FINAL-MAA.cif), and a nearly complete model (working_macrocycle.pdb).

Analyzing Diffraction Data Quality using Xtriage

Open Phenix and select 'New Project'. Name the project "Crystallography Exercise". Do not worry about a sequence file, it is not needed to run the program. Select Xtraige from the 'Reflection Tools' menu (shown below).
Xtriage Select
After selecting Xtraige you should see the following window.
Xtriage Input
Select the data_set_merged.mtz for the 'Reflections file'. This will propoagate the Unit Cell and Space Group automatically. Select I(+), SigI(+), I(-), SigI(-) in the 'Data Labels' drop down menu. Enter 16 residues for the total number of residues in the macrocycle. Hit 'Run' at the top of the menu. After the run is complete you will see the following information:
Check the 'Data Quality' and 'Twinning analysis' tabs to ensure that the spacegroup was processed correctly. The spacegroup for this data set is R 3 2.

Finding the Anomalous Atoms

The data set contains an anomalous signal from an Iodine atom on a single iodo-phenylalanine. Hyss (hybrid substructure search) will find the location of the iodine atom in the crystal lattice. Go back to the main Phenix window and select 'Hybrid Substructure Search' under the 'Experimental Phasing' tab.
Hyss Open
Hyss Setup
To setup a Hyss run you will need to provide a reflections file (data_set_merged.mtz) and enter the wavelength used during the experiment (1.54 Å), the number of anomalous groups (1) and the scatter type (I). Click 'Run' at the top of the program to begin the search. When the search is complete you should see the following screen:
Hyss Complete
The Correlation Coefficient is an important value to note. Data sets that have values greater than 0.3 will more than likely generate solvable electron density maps. For correlation values lower than 0.3, try changing the number of scattering atoms to a lower or higher value than your original guess. Click on 'Run Autosol' to begin the electron density map generation.

Creating a Density Map Using Autosol

If you clicked on 'Run Autosol' from the Hyss window (see above) then all of the files you will need have been transfered automatically to the Autosol window.
Autosol Start
Select I(+), SigI(+), I(-), SigI (-) for 'Labels'. Enter '1.54' for the wavelength of the experiment. Click on 'Guess missing f' and f" ' to have Autosol guess the f' and f'' for the iodine at 1.54 Å. Unclick the 'Autobuild model' box. Click 'Run' to start the program.
Autosol Finish
When Autosol is complete check the 'FOM' (figure of merit) value. A value higher than 0.6 will usually correspond to a decent to good map. Also check the BAYES-CC value. A BAYES-CC value higher than 50 is also a sign of a good map.

Placing the Model in the Density Map

We are now ready to place the working_model.pdb into the density map. Before that we need to acclimate ourselves with Coot, the main program we will use for model placement and manipulation. Open Coot or WinCoot. Select 'Auto Open MTZ' from the 'File' menu and find the 'overall_best_denmod_map_coeffs.mtz' in the Autosol_X folder you just ran. A density map should appear in Coot.
Coot Start
We now want to load the model in the density map. Choose ' Open Coordinates' from the 'File' menu and then find the 'working_model.pdb' that came in the data ZIP file you downloaded previously. This should place the model directly into the density and center on the model.
Coot Start 2

Adjusting the Model to the Density Map