[ccp4bb] Predefining origin for molecular replacement in polar space group
Dear all, Is there a way to specify (somewhat arbitrarily) a origin for any molecular replacement package (in a polar space group) without resorting to phased translation so that one could more easily compare results from different MR runs? Thanks, Shao-Yang -- Shao-Yang Ku EIPOD Post-doctoral fellow Schneider Group EMBL c/o DESY Notkestr. 85 22603 Hamburgphone: 0049-(0)40-89902-160 Germany email: s...@embl-hamburg.de
Re: [ccp4bb] degradation of protein durring freez thaw
From your description, the protein concentration dropped from 10mg/mL to 1-2mg/mL after freeze-thaw. It's hard to imagine that your protein has been degraded. Degraded by what (protease)? At -80oC? Did you see a ladder of lower bands on the gel after the freeze-thaw? If you're interested in anecdote, a protein I used to work on would degrade at 4oC (but in 2 days, not overnight) and precipitate (after thawing) when snap-frozen in liquid nitrogen unless there is at least 25% glycerol in the buffer. Good Luck, SY Quoting Jhon Thomas jhon1.tho...@gmail.com: Hello BB I apolozize an off topic query. I am working with small proetin-protein complex of 24kDa. I purify this N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, 0.3M NaCl . After purification this protein complex are dialysed in 20mM tris pH=8.0.I am able to purify enough amount of protein for crystallisation, which can be concentrated upto 10mg per ml. Then i check the dgradation on the polyacrylamide gel after concentration of the protein. I donot see any degdation protein band on the gel. I store the protein at -80 in aliquotes of 100ul immedaitely after concentration in same buffer. protein concentartion are done at 4 degree by centrifugation. Next day before setting up the trays for crystallisation screening, protein solution concentration check is being done. it turns out that this complex has degraded and concentration is only 1-2 mg per ml. i would appreciate the suggestions to prevent the degradation of complex or How should i make it more stable? so, that i can proceed for the crystallisation. I would really appreciate the suggestions. Thanks in advance Thomas
Re: [ccp4bb] Acta E
Quoting Bernhard Rupp b...@ruppweb.org: I suppose most have read the Acta D editorial already http://journals.iucr.org/d/issues/2010/01/00/me0408/me0408.pdf but it seems another creative way of structure generation has been discovered by some small molecule people: http://journals.iucr.org/e/issues/2010/01/00/me0406/me0406.pdf http://www.china.org.cn/china/2009-12/31/content_19161509.htm The university on Tuesday fired the two authors and asked Zhong to return 32,000 yuan ($4,700) in incentives awarded by the university, 10 days after the journal published a notice to delete their papers. Swift action in China. Best, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@qedlife.com bernhardr...@sbcglobal.net http://www.ruppweb.org/ - The hard part about playing chicken is to know when to flinch -
Re: [ccp4bb] small molecule library
informahealthcare.com/doi/pdf/10.1517/17460441.1.1.1 Quoting c.weinert c.wein...@bioc.uzh.ch: Dear all. I am looking for a library of small (biologically relevant) compounds to test binding to a protein. Does somebody know, if there is a company that sells such a library for fragment based library screening? (to a reasonable price). And does anybody has experience with screening such libraries? I assume that one approach would be soaking the protein with the compounds, followed by SEC and MS analysis. Anybody tried directly soaking crystals followed by X-ray analysis? Thanks alot already in advance for your help. Sincerely, Christopher -- Shao-Yang Ku EIPOD Post-doctoral fellow Schneider Group EMBL c/o DESY Notkestr. 85 22603 Hamburgphone: 0049-(0)40-89902-160 Germany email: s...@embl-hamburg.de
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] soaking with Samarium chloride
Hi Amit, I wouldn't worry about acetate depleting the Sm3+. I have tried to make a derivative of a kinase by soaking ions of lanthanoid series but in the presence of phosphate buffer and ADP. In this case, lanthanoid ions are *known* to precipitate with phosphate (and phosphoryl moiety). I simply spun down the precipitate and took the clear but colorful supernatant for soaking. A successful ADP-2Ho derivative for phasing is reported in Acta D 2007 63:493-9. (Failed cases were rejected by JFCE on April 2.) Shao-Yang Quoting amit sharma 3112a...@gmail.com: Dear All, I have a crystal growing in the presence of 0.1M Sodium Acetate pH 5.0, 10% PEG4000, 7.5% Dioxane and 10% Ethylene Glycol. I wanted to know if it would be alright to use Samarium chloride for derivatization. I am worried about the leaching of samarium by acetate. Also, what soak times can I use and what conc. of Samarium chloride? My protein is 10 kDa , has no cysteines/methionines and pI for the molecule is 4.6. Also, could somebody suggest what other heavy atom soaks could be performed in this case? Any suggestions would be appreciated. Many thanks in advance -- Amit Sharma
