This post has a question and an answer (good karma)...answer first In response to Matt's question about periplasmic harvesting, we do an osmotic lysis of e.coli by a series of centrifugations. First, harvest your cells normally (4k 15 min). Then vigorously resuspend in a 20% (w/v) sucrose, 1 mM EDTA + buffer (we use 20 mM tris). My best yields are 1-2 mL sucrose buffer per 1 mg of protein you expect to get. Equilibrate at RT ~20 min before centrifuging 8k 15 min. Then resuspend the pellets in 5 mM MgSO4 same vol ratio as before (should see foam if you did it right). Finally, spin that down 9k 15 min and collect your periplasmic lysis. You'll want to adjust the pH of the product, depending on what column you want to purify, ours comes out <7 and needs to be >7 for Q sepharose. Enjoy!
And my question: I have 3 distinct NCS pairs in the structure I'm refining, and I'd like to make my job a bit easier by applying the NCS edit function in COOT after a round of tinkering. However, when I go to type in the command: copy-from-ncs-master-to-others imol master-chain-id, COOT just sits there like it doesn't know what I'm talking about. Maybe I'm not telling it the proper syntax for master-chain-id? I'm using COOT 0.5.2. 'Preciate it, Geoff -- Geoffrey K. Feld College of Chemistry University of California, Berkeley "Vigilia pretium libertatis"
