We will begin where the previous practical finished, by inspecting a CD44 model which has been automatically built by the program Buccaneer. The data for this tutorial may be found at cd Run the task. If you are prompted about nomenclature errors, just click Yes. Scroll to a value near 1.
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Go to the last table in the Tables in File and click on: Rfactor analysis, stats vs cycle You will see a graph of the R factor and the Free R factor for the 6 cycles of refinement. The R factor is very good already but both go down a little. Also, slightly up the Tables in File list, select the last: Cycle 6. Rfactor analysis, F distribution v resln This is information about the last cycle of refinement.
This is similar across the resolution ranges - it does not go up for high resolution data. This is an example of what is good about maximum likelihood refinement compared with the old-fashioned least squares.
You notice that at low resolution the observed red and the calculated blue are not the same. At low resolution the water atoms, which we can not see in the crystal structure, are an important part of the structure factors. The refinement program tries to model the water atoms by solvent scaling but it is difficult for this data because some of the very low resolution data is missing.
To close the loggraph window click on the File menu and select Exit. The DELFWT map is the weighted difference map of F observed - F calculated and looks like this: Here you can see a large pink area of negative density where the tyrosine side chain is now.
This is saying that the side chain should not be here. The large brown-red area of positive density is showing where the side chain should be. The FWT map is the weighted map and looks like this: You can see region of density to the left of the tyrosine which is where it should go. In fact there is a full monomer description of the ligand in our example in the library, but that is too easy.
We will just have a look at it. If you have coordinates for the ligand in a PDB file, it is possible to make a monomer library entry very quickly - we will try doing this. If you have no coordinates for the ligand you need to draw the molecule after which the programs will make a geometry description and will also make a PDB file with coordinates. This can be made easier if there is a similar molecule in the library - you can get this molecule from the library and edit - we will also try doing this.
You will see the molecule displayed. You can rotate it by holding down the left mouse button. On the right of the window is a list of atoms - this list has the element, the atom name and the oxidation state the charge of the atom. Below the list of atoms is a list of the chiral centres found in the molecule, of which there are four. Now look at the monomer library file. In this file you will see a list of the atoms. The refinement program will try to make the ligand as defined in this file - you can edit the file if you need to.
If you already have sketcher open from the previous exercise, delete any molecule that you have displayed: from the Edit pull down menu select Delete All Atoms. The picture of GMP below shows the correct delocalised and aromatic bonds - edit your molecule accordingly.
To change a bond to a delocalised bond, you must hold down the Shift key on the keyboard and click on the bond with the right mouse button. It will step through single--double--triple--deloc--aromatic--metal.
Now create the monomer library. From the File pull-down menu at the top of the window select Create Library Description. Then Close this window. When it has finished the molecule is displayed again. Now look at the new monomer library file. From the Main Window select the last job which is called dictionary. This list is not quite the same as for the monomer 3GP as it is in the Monomer Library.
Have a look at the differences and update as you see fit. Alternatively you can go directly to the next step - Review Special Restraints for ligand. If you have no coordinates or other definition of the ligand then you must draw the molecule in the Sketcher. Sometimes there may be a similar molecule in the library - you can start from this and edit it. Delete any molecule that you have displayed: from the Edit pull down menu select Delete All Atoms. You must wait a little while before the molecule of guanosine is displayed.
The atoms that you add will be carbon atoms - you will change them later to phosphorus and oxygen. It is now the flashing, active atom. You now have an atom called C21 and it is the active atom. Click close to this atom with Shift - left mouse button to make one more atom. Add two more atoms. You will need to make the C21 atom the active atom Control - right mouse button and then add the atom for each of those. When you have finished adding atoms, click on the Do nothing edit tool at the top left of the window; now you will not make more atoms by mistake.
Now look at the end of the table on the right side of the Sketcher window. Change C21 to a P and the other three to O. Now we create the monomer library. Close this window. Wait while the program runs to build the dictionary file. The molecule is drawn again. If necessary you can make corrections and run again. To close the Sketcher window, select the File pull-down menu and Close Sketcher.
The program will look at the atom coordinates again. The disulphide bonds, cis-peptides and D-peptides are already defined. Of course this is not always right. Exercise From the Refinement module select Run Refmac5. From the Run menu at the bottom of the window choose Run Now.
If all is well, the program should run without any warnings, apart from those about hydrogens. Now we are ready for some real refinement. Enter a suitable job title such as Job title restrained refinement for liganded RNAse refine tutorial step Then Do restrained refinement using no prior phase information input Also you will see: Generate weighted difference maps in CCP4 format.
If you have a graphics program to look at the maps then click this on and select a map format.
CCP4 Tutorial: Session 1 - Introduction
If you have problems following the instructions, then you can use. Often you will use the output file of one job as the input file for the next job. However, if you do not have the output file, then it will also be available in directory DATA. You also need to define directories so that ccp4i knows where to find files.
CCP4 Tutorial: Session 6 - Refinement
CCP4 Tutorial: Contents