Hi all, Here's the situation: We have used a 30% PEG8000 and 20% PEG400 mix to stabilize our crystals and to act as cryoprotectant. We soaked in a pentasaccharide substrate into the crystal (or at least we added the substrate to the soak solution and allowed the crystal to soak several hours) and collected data at -100C. The maps do show partial ring structures in the binding site, but the occupancy is very low and there is no connectivity. In the spaces between protein molecules, we see long stretches of connectivity. Questions: 1) Is the long connectivity PEG400? 2) Since PEG400 is at 20%, can PEG400 prevent the saccharide substrate from soaking into the crystal since the percentage of saccharide is much lower than the percentage of PEG400? 3) Has anyone had similar experiences seeing PEG400 in their maps? If so, do you have a structure/parameter/tophology of PEG400? 4) Can we use the substrate itself as a stabilizer and cryoprotectant to replace PEG400? Say use a polymer of the substrate at about the same molecular weight and at the same percentage? 5) Other than sucrose, is there any other disaccharide which has been shown to be a good cryoprotectant? Any and all help, tricks, comments (good or bad) and suggestions are welcomed. -- Robert D Scavetta Dr. Frances Jurnak's Laboratory Department of Biochemistry 1447 Boyce Hall University of California Riverside, CA 92521 email: scavetta@xtreme.ucr.edu phone: (909) 787-4196 fax: (909) 787-3590
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