Re: [ccp4bb] Calcium soaking
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 unn...@mx.ibaraki.ac.jpwrote: 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
[ccp4bb] Calcium soaking
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
Re: [ccp4bb] Calcium soaking
Masaki, If your crystals crack when you add calcium it implies that calcium binding induces a conformational change. You should try co-crystallization with an epitaxial jump approach: Stura, E. A., Charbonnier, J.-B. Taussig, M. J.(1999) Epitaxial jumps. J. Cryst. Growth 196: 250-260. Since it is likely that calcium binding destroys only one crystal contact, you should screen for co-crystals with seeds of your calcium-free crystals. One of the planes of your calcium-free form should be able to stimulate growth of crystals with calcium but your crystallization conditions are likely to be different. Without seeding you are likely to be hindered by the nucleation barrier which seeding overcomes. Enrico. On Thu, 20 Feb 2014 11:29:47 +0100, Masaki UNNO unn...@mx.ibaraki.ac.jp 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 -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
