Aim: we will fit a domain of the Cleavage and Polyadenylation Factor
Using a web browser, download the bundle for EMD-3908
Move that tar file here and extract it.
Let’s download the sequence of a fragment of the structure:
Move that sequence file to the current directory (for easy access).
$ coot --map EMD-3908/map/emd_3908.map
Let’s see more of the map
Let’s use smoother maps
Compare these maps and then delete the 1st (mrc) map.
Choose the FoutBlur_200.0 map for fitting (at the moment):
Change the contour Step for the new Maps:
Display Manager → Properties → Change by rmsd
0.33 → OK
As a rule of thumb, a good contour level is 5.5 rmsd, but for the blurred map we should use about 0.03 V (9.4 rmsd).
Move back to the middle of the map with Undo Navigation:
Unless you’ve moved the view around, you should be at the centre of the map: (112, 112, 112). If the centre has moved, you might need a bit of manual assistance:
It should roughly fit now. If it doesn’t, try jiggling again once, twice or perhaps several times (either that or you didn’t follow the instructions 😄).
In this case, you should be looking for a fit score of over 1000.
Extract the worst-fitting (WD40) domain:
Let’s work on this fragment:
Let’s delete the sidechains from the atom selection molecule (number 4 usually):
For the most recent model, use
We need to adjust the weighting of the map to the model:
Let’s add some local distance restraints:
When the refinement dialog says “Success,” examine the model, being careful not to inadvertently pull on an atom. Maybe you will see that there is a domain that doesn’t fit, if so, yank on the worst fitting CA and pull it to where you think it should go.
When you are happy, dismiss the Refinement dialog:
It can be non-trivial to decide what needs to move and how to move it. It is worth undoing your modifications and refining again for practice.
This time perhaps without drawing the restraints:
… or with different cut-off for the Geman-McClure restraints, or a different alpha for the Geman-McClure restraints, or a different weight for the map. Or a different blur for the map. You can delete the current extra restraints with ProSMART → Delete All Extra Restraints.
Reset Geman-McClure alpha to 1.
Upon review, you will notice that there are parts of the model that
don’t fit the map. Try yanking them around with Tandem Refine. Other
parts of the model don’t have density - so delete the residue range - this will help the alignment we are about to do.
Maybe the density fit validation dialog will be useful? You will need
to reset the weight:
print_sequence(4)Note: the molecule number that was a result of “Copy Fragment”
associate_pir_alignment_from_file(4, "A", "a1-align.fasta")Note: use the molecule number that was a result of “Copy Fragment”
(Note: this interface will change in the near future)
Go through the structure residue by residue looking for things to fix
There will be places where you need to close (or open) a loop by renumbering residues.
Apart from the alignment in Section 8, you can, with a bit of practice, comfortably do both of these tutorials in less than half an hour. You may have some experience with Coot and now have got this far, so try to refine (with your favourite macromolecular refinement program) the domain that you have just modelled. Use validation and fix up some issues. You can use Coot’s validation analysis:
(validation-outliers-dialog 4 1)
After that, you can try another domain (I suggest the “B” chain).