Tutorial :
fitting residues into electron map density
In this example, we will learn how to guess
what peptide fragment would best fit into the density, given
a poly-Ala and the sequence of the protein to solve.
Step by Step
open file pdb file 1LDB_ala
(provided with the tutorial
package).
open the dn6 map 1LDB.dn6, and set the following
parameters into the dialog: Show unit Cell, display around
CA with 8.0 Å around x y and z, and contour at 0.8
sigma (the fitting works better when ample space within the
density is given to accomodate residues, and a contouring
value below 1 sigma is usually recommended).
Load raw sequence of the protein to solve (1LDB.txt) from
the SwissModel menu and display the Layers Infos Window
(from the display menu). Remove the visible checkmark for
the layer 1LDB.txt as we need it only to have the sequence
of the protein to solve.
Enable the slab mode (from the Display menu) and center
the protein on the Ala63 Alpha Carbon by option clicking
(right mouse button for PC) on Ala63 in the control panel.
Select Ala61 to Ala65 using the control Panel, and use
the "Find best fitting peptides item of the Build menu". Be
a little patient, and a result file will be opened:
DAMDF mismatch: 0 In: 19 Out: 1 Score: 11.20
LARAF mismatch: 0 In: 18 Out: 2 Score: 8.57
KGATY mismatch: 0 In: 15 Out: 2 Score: 8.54
RAILH mismatch: 0 In: 18 Out: 4 Score: 8.31
ERDVY mismatch: 0 In: 21 Out: 6 Score: 8.15
DILTY mismatch: 1 In: 18 Out: 5 Score: 7.89
The files gives you the list of existing pentapeptides
that would fit onto the backbone fragment you selected, the
number of residues that do not match well, the number of
atoms inside and outside the density, and a score value.
Results are sorted by score, but you should also consider
the other parameters when looking for the best solution (and
of course rotate the protein and actually try the
various solutions.
Click on the first line to have a look at the solution.
Explore the various best solutions (you can either click on
a solution, or use the up and down arrows while the text
window is active. Be careful, because the up and down arrows
will have the efect of changing the sigma contouring value
when the Text window is not active.
Now display the Align window and compare your solution
with the actual sequence of the solved protein to look
whether you guessed right (you could have a view like this
one in stereo).
Check that the sigma contouring value is still set to
0.8, else set it to 0.8. Now center the protein on Ala35,
select residues Ala32 to Ala37, hit the return key to
display only this section of the protein, and use the "Find
best fitting peptides" again.
ANESK mismatch: 0 In: 15 Out: 2 Score: 8.91
KNRFH mismatch: 1 In: 20 Out: 9 Score: 8.71
ARFRF mismatch: 0 In: 22 Out: 7 Score: 8.64
RIFVN mismatch: 0 In: 20 Out: 5 Score: 8.24
TNPVD mismatch: 1 In: 17 Out: 4 Score: 7.98
GARVV mismatch: 0 In: 11 Out: 3 Score: 7.67
MDFNH mismatch: 1 In: 19 Out: 6 Score: 7.44
ASYVF mismatch: 0 In: 16 Out: 5 Score: 7.27
AIFRS mismatch: 0 In: 17 Out: 4 Score: 7.20
PKPVD mismatch: 1 In: 17 Out: 5 Score: 7.13
CRDAD mismatch: 1 In: 15 Out: 3 Score: 7.13
Again, try to guess what is the best fragment. This
illustrate that the top ranking fragment is not always the
best one, but usually, the best one is among the top ones.
Now repeat the process for residues Ala38 to Ala42.
And finally, repeat the process for residues Ala43 to
Ala47. Surprise: this time the correct fragment is not among
the top ranking ones. Let's see why. Select residues 43 to
46 only, and find the best fragments. This time, the correct
solution is among the top ranking fragments. So what happens
with residue 47 ?
Click on the mutate tool in the main display window, and
then click on residue 47. Mutate to Glu, and browse the
rotamer library. As you can see, no rotamer fits even
approximately into the density. Select rotamer 1, and accept
the mutation. Now click on the torsion tool, and select
residue 47. Use the little arrows that have appeared at the
right of the torsion tool to alter the chi-1 and chi-2
angles of the rotamer until it fits into the density.
Now, reset the orientation, load 1LDB.pdb and compare
your fit with the actual solved structure.
This ends this tutorial section.
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