Have you tried lower concentrations of Calcium soaking untli the crystals do not crack? Or does it crack even at very minute calcium concentration?
On Thu, Feb 20, 2014 at 3:29 AM, Masaki UNNO <[email protected]>wrote: > Dear all > > Apologies for the off-topic question: > We are studying an enzyme that is activated by Ca2+. We obtained the > crystals of the substrate and Ca2+-free form and solved the structure at > 2.7 > A resolution. However, the active site electron density map was not clear, > although other regions are clear. We would like to determine the > substrate-complex with Ca2+, which will elucidate the active site structure > at a higher resolution. > Now we have a problem that the crystals of a mutant which can bind the > substrate and Ca2+ always have cracks in soaking to the crystallization > solution containing CaCl2. Co-crystallization does not work at this time. > We > estimate the structural change in Ca2+-binding is not so big because the > isozyme structure did not change very much when binding Ca2+. An isozyme > structure in complex with the substrate was determined by soaking Ca2+ (and > the substrate). > > How should we overcome this problem? > > Best regards > > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ > Masaki UNNO > > Graduate School of Science and Engineering, Ibaraki University, Japan >
