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. 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
