Hi Tiancen,
provided that you know for sure that your Gly->Pro mutation has been
incorporated (see Artem's comment) I would spend some more time
modeling your Pro.
Here are some things to consider:
(1) If the view of the model you provided has not fooled me, I
believe that the main-chain model for your Proline is quite atypical
for prolines. I estimate something like phi=-90 and psi=-10. That
would of course be just fine for a GLY. Did you model the Proline
according to evidence in difference electron density maps, or did you
just mutate your Glycine using a graphics program and just let REFMAC
push it in the 'right' place. Of course at 3.3 angs resolution you
cannot expect much on that front. What does your Ramachandran plot
look like for the mutant and the wild-type structures at this position?
(2) Given that this is a rather drastic mutation, it may just be that
the PRO side-chain is behaving suboptimally and is simply
flip-flopping between the preferred chi-1 angles of +/- 30 deg. This
could explain the absence of density around the C-gamma position.
(3) I agree with Bill Scott's suggestion to model the cis-Pro as well.
There is a well known variant of Ribonuclease A, the P114G (Schultz et
al 2005), in which a cis-Pro is mutated to a Gly which in turn adopts
the trans configuration. Configurations aside, this the opposite
mutation to yours, but I could not refrain hallucinating about a
possible reversal of scenarios...:), i.e transGLY->cisPRO.
Best wishes
Savvas
----
Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html
Quoting HTC <[EMAIL PROTECTED]>:
Dear all,
Recently, I crystallized a mutant protein in which a Glycine was changed
to Proline. The resolution of the crystal is 3.35A. I performed molecular
replacement and refinement with CCP4. Surprisingly, no electronic density
can be detected for the side chain of Proline. Since Proline is always
considered rigid, I wonder if it is possible that this Proline is so
flexible that its map becomes undetectable? The density map is attached.
Thanks in advance!
Tiancen Hu
Shanghai Institute of Materia Medica
Rm. 2107, #555, ZuChongzhi Rd.
Shanghai 201203
P.R. China
