On Wednesday 16 September 2009 11:19:11 Ivan Shabalin wrote:
> Dear all,
>
> I have an atomic resolution (1.1Å) structure of enzyme with the bound
> cofactor NAD. During the analysis of the refined structure I found that
> important double C=O bond of the cofactor in the active site was slightly
> lengthened from standard 1.22Å to 1.26Å.
I suggest that if an important aim of your experiment is to set accuracy limits
for the length of a particular bond, then you should use full-matrix refinement
in shelxl and have it specifically calculate the standard errors.
This will be a more accurate determination than anything based on global
properties, such as the DPI.
cheers,
Ethan
> Then I increased the sigma's of the
> distance restraint in the REFMAC dictionary 50-fold and as a result the
> distance increased to 1.29 Å. I suggested that this deviation from
> standard
> double C=O bond means decrease in the bond order to something between 1 and
> 2. This change in bond order upon cofactor binding (and especially in the
> transition state of the reaction) was suggested previously by studies of the
> kinetic isotope effects and partly confirmed by quantum mechanics
> calculations.
>
> This change in 0.07Å is quite small, but is a bit higher than the DPI
> multiplied by a factor of two (2*0.028=0.056). Is it crystallographically
> sound to state the bond lengthening? Is DPI the correct estimation of error
> of positional parameters in my case (note that B-factors in the active site
> are about 12-15, when average B-factor for all atoms is 20)? Maybe, ML based
> su of positional parameters is better estimation.
>
> Statistics of the dataset (values in parentheses are for the
> highest-resolution shell 1.11-1.10 Å):
> R-merge = 5.1 (52)%
> Redundancy = 4.0 (2.5),
> Completeness = 96.5 (73.8)%
> <I>/<σ(I)> =44 (1.9),
> mosaicity = 0.7 (I think, this value is a little bit high).
>
> Statistics of the refinement with REFMAC:
> R-factor =13.4%
> R-free =15.9%,
> RMSD_BOND = 0.015Å
> DPI = 0.028
> ML based su of positional parameters = 0.0217.
> Estimated with SFCHECK maximal error 0.062 A
> Estimated with SFCHECK minimal error 0.014 A
>
> All help greatly appreciated!!
>
> Yours,
> Ivan
>
--
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742
