> > Dear ccp4bbers, > > > I need some suggestions regarding structure solution of fused protein > complex. > > After repeatedly failing to obtain crystals of a 45 kDa protein (mostly > a-helical) we fused the protein of interest to another protein of known > structure which is 30 kDa is size (mostly b-strands). Many fusions were made > and we selected a fusion-protein (*Ca*. 75 kDa) that has a minimal linker > between the two proteins for crystallization trials. Both partners in the > fusion are active and function as expected of wild-type proteins. > > > > Crystals of the fusion protein were obtained and couple of datasets was > collected. Running crystals of the fusion protein after X-ray data > collection on SDS-PAGE shows that we have a band that is running approx. 60 > kDa instead of 75 kDa indicating some form of proteolytic processing during > crystallization (there are no bands that are equal to the 30 kDa fusion > partner of known structure). One of the crystals diffracted to 2.5Å > resolution. We could index the data in P2 (as well as P222 and P212121) > space group, the highest resolution shell was cut off at 2.5 Å using I/σ = > 2.0 as a guide. The overall Rmerge of 15% and completeness of 99%. The > Matthews coeff. is 2.32 with 43% of solvent content as calculated with the > molecular wt. of the fused protein complex (75kDa). We used Phaser (AutoMR, > Phenix) and Molrep and Amore (CCP4) for molecular replacement using the > known structure as the search model (the 30 kDa protein). In P222 and > P212121, the statistics after one round of refinement post-MR statistics > are good (R-factor and R-Free of 21% and 24%, respectively) and the known > protein molecule fits and packs well with no extra density for the protein > of interest. In contrast, in P2, we can see the extra density between planes > formed by the known structural model. The spacing between these planes is > about 30Å. The statistics of one round of refinement post MR is very poor > (R-factor and R-Free of 41% and 47%, respectively). > > > The questions we are interested in are:- > > 1) How do we determine the correct MR solution (P2 or P212121)? > > 2) How should we proceed further in case like this? > > > > Thanks in advance for the suggestions. > Bob
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