My protein of interest, PEX19, is farnesylated at its CTD. My goal is to mutate residue 255 and determine if there is loss of polar contacts between the new amino acid and the farnesyl moiety. For some reason, PyMol just won't let me show polar contacts with the farnesyl. Am I missing something?
The farnesyl group is apolar ( https://pubchem.ncbi.nlm.nih.gov/compound/6438372#section=2D-Structure ) and linked to the cysteine via a thioether linkage, the only polar interactions would involve the peptide bonds linking the farnesylated Cys to the adjacent amino acids ( https://www.nature.com/articles/ncomms14635 ). Farnesylation of proteins usually serves as a membrane anchor
Annemarie Honegger thanks for your helpful answer. I understand that the farnesyl is linked to the cysteine in the CaaX box of the PEX19 protein (https://www.nature.com/articles/ncomms14635), but did not realize farnesyl is apolar. According to this paper, reside M255 (along with several other residues) in PEX19 forms part of the hydrophobic farnesyl binding pocket. Formation of this pocket containing farnesyl is crucial to forming cargo binding sites on the surface of the PEX19 protein. Mutation of this residue to an Arg leads to a weakened protein-farnesyl interaction within this hydrophobic pocket. Would it somehow be possible to show this weakened interaction between M255R and the farnesyl moiety using PyMol?
Paste this into the PyMOL command line:
fetch 5lnf
select FAR, /5lnf/B/A/FAR
select FARcontacts, br.((5lnf within 3.0 of FAR) and not FAR)
show sticks, FARcontacts
hide sticks, hydrogen
color green, 5lnf
util.cbac FAR
util.cbas FARcontacts
color red, /5lnf/A/A/MET`255/
zoom FARcontacts
set_view (\
0.938094318, -0.337558001, -0.077626705,\
0.172650948, 0.261420399, 0.949657857,\
-0.300272107, -0.904274464, 0.303518116,\
0.000000000, 0.000000000, -62.998535156,\
0.883457184, 1.198143959, 0.834658623,\
49.668525696, 76.328544617, -20.000000000 )
The farnesyl moiety is shown in cyan, residues with at least one atom within VdW contact distance of it in salmon, Met 255 in red. You can clearly see that by replacing Met with Arg (and associated water molecules), it will obstruct the Farnesyl binding pocket
Dear Yasmin D'Souza I found your question quite fascinating, perhaps because as a synthetic inorganic chemist I have absolutely no idea of how anyone can do and understand such calculations. Thus in addition to the absolutely perfect answer provided by Annemarie Honegger there isn't much that I could add. The only thing that is easy for me to see is that the farnesyl group is an unpolar long chain hydrocarbon substituent which is rather flexible. Rotation is possible at each of the C–C single bonds in this group. I found it interesting to see that farnesylation of your protein of interest has already been reported in 2009:
Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis
Article Farnesylation of Pex19p Is Required for Its Structural Integ...
Let me also suggest the following detailed study (PhD thesis) which might be of interest to you (if you don't know it already):
Structural characterisation of peroxisomal import receptor complexes
https://bib-pubdb1.desy.de/record/448369/files/Structural%20characterisation%20of%20peroxisomal%20import%20receptor%20complexes.pdf
(see attached pdf file)
Please keep on asking such interesting questions on RG! Good luck with your research and best wishes, Frank Edelmann
Thank you Annemarie Honegger for your help. Your answer is great. It is just too bad that I cannot visualize more clearly the loss of interactions (loss of dotted lines) with the mutated residue.
Thank you Frank T. Edelmann for the links you posted. The thesis was a very interesting and helpful read!
You can use the "distance" command ( https://pymolwiki.org/index.php/Distance )
distance [ name [, selection1 [, selection2 [, cutoff [, mode ]]]]]
e.g
distance metCE, /5lnf/A/A/255/CE, (5lnf and not resi 255), 3.0, 0
to show distance vectors from the Met CE to all atoms within 3.0 Å.
if you change the last parameter to 2, only polar contact distances are shown, (none in this case)
Annemarie Honegger This is exactly my issue. The command you describe is not working for me:
distance metCE, /5lnf/A/A/255/CE, (5lnf and not resi 255), 3.0, 0
I do not see any vectors with this command. Would you kindly try it in PyMOL and paste an image of the result please? Thank you so much for your help!@
Which version of PyMOL are you using? This Image was generated using PyMOL 2.4, the script I sent before and the distance command listed above.
You can increase the distance cut-off to see more contacts, in particular to see the heavy-atom - heavy atom distances rather than the distances to the nearest hydrogens.
I am using PyMOLv 2.5.1 - Incentive Product. This is the 30 day free trial for mac users. Perhaps this is why it is not working for me.
No, that is not the reason - I have tested it on PyMOL 2.5.1 on my other computer, and it works as well there, copy-pasting the scripts from this thread.
Do you see the distance object "metCE" listed in the object window? Do you get any error messages in the feedback window?
In the feedback window it says: Executive: object "metCE" created. The object menu is completely blank. There are no vectors on the structure. However I have managed to visualize this by using the wizard-->measurement function.
??? - You should at least see the molecule object (5lnf) in the object window, as well as the two selections (FAR) and (FARcontacts) generated by the first script. After running the distance command, metCE should appear as a new entry, allowing you to display or hide the object by clicking on it or change its color. in the C pulldown.
Did you enter the command manually, or copy-paste it from here? You may want to try to increase the distance cut-off from 3.0 to 5.0.
You can of course use the distance wizard, but this requires you to select each pair of atoms individually rather than automatically determine which atoms are within range.
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