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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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. Automatic model building will only rarely produce a model which is both complete and correct. Then choose to link chain A and the file cd Since Buccaneer has built an Ala residue for any unknown residues not docked with the protein sequence during model building and marked them UNK there is no need to add any atoms at this point.
It should be quite clear where the side chain of Asn 21 should be placed. The small loop from A 22 to A 25 has proved more difficult to build automatically it looks like auto-tracing has followed a side-chain rather than the main-chain at one point.
Fortunately, the map in this region look quite good so we will try to complete this region of the model using the loop fitting tools in coot. Check the fit of residue Asn 21, paying special attention to the position of its carbonyl oxygen. Enter residue numbers 22 and 25 as the beginning and end of the region to be built. The model now looks like it fits the map a great deal better, but there is still some room for improvement so we will carry interactively refine the region we have just built.
Are you happy with the position the putative refined residues have adopted? Do the traffic light indicators all show green, signifying good model geometry? If so, accept the refinement and continue. If not, try to improve the model, perhaps with help from a demonstrator. To me, it looks like residue A 93 has been somewhat misplaced but residues A are placed well in the map.
We can trust their placement in this density sufficiently to trust that there are no residues incorrectly missing or added and we can therefore extrapolate the sequence back from A 97 Asp.
Coot allows us to mutate a range of residues at once, and can attempt to fit the newly placed sidechains in density automatically. Tick the box to Autofit mutated residues. It is usually a good idea to extend the refinement one or two residues beyond the region you have just built, so in this case residues 94 and 97 would be good beginning and ending points.
We are left with a model where residues A 92 and A 93 have not been built. Looking at the map in this region it is very difficult to see where the main chain should be traced, so we are better off leaving the residues absent. With luck, future refinement will improve the map sufficiently to allow these residues to be built. There does not seem to be any density to support adding any more residues to the C-terminus of the current model.
Work your way along the polypeptide backbone inspecting the fit between the model and the electron density map. When you reach residue Asp 5 you will notice that the model fits the map very poorly. Use the Auto-Rotamer tool to correct the orientation of Asp 5.
When you reach residue Arg 11 you will notice that the model fits the map poorly. You will notice that the resultant model is still a rather poor fit with the electron density map. Fortunately it is possible to intervene manually in cases such as this where the refinement has become trapped in a false minimum.
Use the mouse to drag the refined model the one with the carbon atoms displayed in white into the electron density. You should find that the Arg sidechain will snap neatly into the map once you have dragged it in the right direction.
As long as you are happy with the geometry of this refined model, accept the refinement. In some cases this can be very helpful. Continue to work your way around chain A, fixing errors where you find them. You may find at least one error that the flip peptide tool will help you fix.
When you have reached the end of chain A or when the demonstrators tell you that you have used enough time on this part of the practical continue to the next point. Buccaneer did not do quite so well building the second copy of cd44 and has split it into two separate chains B and C.
Chain B contains most of the model, consisting of residues Although they may contain some small differences, at this early stage of refinement it is reasonable to assume that the two chains are at least similar to each other, so we can use coot to copy our edited and improved chain A to provide a good approximation of the second molecule. First we want to remove chain C, since it will be in the way. Zoom out by dragging upwards with the right mouse button until you can see all of chain C it consists of two beta-strands joined by a hairpin.
Shift-left clicking on a residue will identify it, so you can make sure that you have correctly identified chain C. Now you can copy chain A onto chain B.
Refinement in Refmac5 We will now use the program refmac5 from the ccp4 suite to refine our corrected model against our reflection data. We will make use of the new NCS tools in the latest version of refmac5. The two new fields that appear can be left at their default values. Run the job. Open the Results tab for job you have just run. This will probably happen automatically.
At the top of this report is a table showing the statistics from the refinement job. The final stats are presented in a table and the change in these values is plotted by refinement cycle.
We would expect both R and Rfree to have fallen during a successful refinement. In addition, R and Rfree should not diverge from each other too greatly - this would be an indicator of over refinement. A difference of approximately 0. It is also important to check that the model resulting from refinement conforms to expected protein geometry.
The summary table at the end of the refmac5 log file lists final values for Bond Length and Bond Angle showing the rmsd from library values. The average rms for these values in the restraint library is listed earlier in the log file - it is 0.
The values for your refined model should be lower than these library averages, ideally substantially lower. If this is not the case, you will need to re-run the refmac5 job. If the geometry is acceptable you can continue to section 4. During a refinement job, refmac5 attempts to optimise the model against two separate targets - the experimentally measured structure factor amplitudes and prior knowledge of protein geometry.
The weighting given to each of these targets during refinement is of critical importance to achieving a successful refinement, and the correct weighting can be very sensitive to data resolution. By default, refmac5 will attempt to automatically determine this weight but it will often require manual intervention for optimisation. Under the Refinement Stats table in the Results tab you were inspecting before you will see reported the weight applied to the X-ray term during the refinement job you have just run.
Enter a value lower than that reported for the last job to tighten the restraints on geometry. I would suggest a possible value of 0.
This value will be very sensitive to both the resolution and quality of your reflection data. A lower value will lead to more tightly restrained geometry whilst a higher value will weight more heavily towards the experimental X-ray terms.
Once again, check the Results tab resulting from your refinement job. Do you think the statistics reported by refmac5 now indicate a more acceptable model? Validation Inspect output from refinement and check quality of the resulting model using validation tools.
When rebuilding and refining a protein model it is very easy to make small mistakes, particularly at low resolution. It is therefore very important to cross-check your protein model against the large body of prior knowledge regarding protein geometry. This is the process of validation. This job is automatically populated with the output from your refinement job, so you can simply Run the job.
Set up the maps and restraints as described in section 2. Exactly what validation and editing are needed at this point will depend both on what editing you carried out in section 2 and exactly how your refinement job was run in section 3.
Here are some suggestions - please note that you are unlikely to have time to fully rebuild and validate this model during the practical session, so try to fix no more than 5 problems using each validation tool in order to get a feel for the tools.
The default sigma level 5. A list of peaks will then be generated - work your way down them, correcting problems as you find them. An interactive Ramachandran plot will be displayed, with any outliers shown in red. Are there any problems in need of attention? Whilst examining the structure, you will have noticed that there are numerous peaks in the electron density map that would be well explained by ordered water molecules.
Coot contains tools to help you add waters to the model. The new waters have been added to chain D, and it is important to inspect them and make sure that they have all been added in appropriate places. Does this water molecule look correct? If not, either move or delete the water molecule as required. Upon correcting as many errors with the model as possible, you would once again save your coordinate model and use it as input for a further round of refinement, repeating successive rounds of rebuilding and refinement until no further errors need to be corrected.
If you have sufficient time, carry out another round of refinement as described in sections 2 and 3. Have the R and Rfree values improved relative to those observed in section 3 as a result of your editing?
CCP4 Tutorial: Session 6 - Refinement
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. You also need to understand how information is arranged in an MTZ file.
CCP4 Tutorial: Session 1 - Introduction