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: [log in to unmask]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html





Quoting HTC <[log in to unmask]>:

> 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
>
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