Re: [ccp4bb] Bfactor is zero?
On 12/20/2010 10:34 AM, Zhibing Lu wrote: Hi All, Recently I solved a structure in which some water molecules have Bfactors at 0 and overall wilson Bfactor is 0.654 based on PHENIX refinement. Is it possible? Bill Lu Hi Bill, What resolution are you working with here? An overall Wilson B 1 is sort of odd... If the resolution is too low - the slope in the Wilson plot has a lot of play - and might be indetermiate I would use phenix.xtriage and see if there are any indications of something interesting happening with your data... Ezra
Re: [ccp4bb] .cv to .mtz conversion
On 9/28/2010 10:41 AM, Jeremiah Farelli wrote: We recently had this problem in our lab. No matter what we tried, we could not get convert2mtz (or any other program) to work properly. We were probably doing something wrong with the fortran? Depending on how far along you are, you can try using phenix.refine. Just input your model and the .cv file and phenix will export a new .cv file with Free-R flags intact. If you aren't to the model building stage yet, this won't work however. Use the program sftools - sorry no GUI... It can handle CNS format - and will convert the test set flag conversion from CNS (TEST=1) to CCP4 (=0) Ezra
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
I cannot say what you have - running a gel, MS, etc of a washed crystal could confirm what you have. What you did not indicate is if your lack of diffraction was of frozen or unfrozen crystals. I have seen too many cases where it is the cryo condition killing diffraction. So if you have not tried yet - try a wet mount. Ezra On 8/30/2010 9:24 PM, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
I cannot say what you have - running a gel, MS, etc of a washed crystal could confirm what you have. What you did not indicate is if your lack of diffraction was of frozen or unfrozen crystals. I have seen too many cases where it is the cryo condition killing diffraction. So if you have not tried yet - try a wet mount. Ezra On 8/30/2010 9:24 PM, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] Query regarding GST fusion protein purification
It sounds like list your GST construct is not binding to the column (or very well) when the peptidase is attached. GST needs to form a dimer to binding to the column - I suspect that your construct interferes with dimer formation - when the peptidase is present, but when not there due to stalled translation (rare codon?) it binds ok. The other possibility is that when your peptidase is present it causes aggregation - preventing binding. Maybe it depends on the concentration of your construct - possibly dilute and slow binding might work - but am not sure. Also - is there much of a linker between the GST and the peptidase? Maybe others have a suggestion... Good luck, Ezra On 8/29/2010 7:28 AM, Ashok Ranjan Nayak wrote: Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased Nacl concentration upto 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40 mM in the elution buffer with two different pH at 8 and 9. I also read from literature that similar intracellular cysteine proteases behave same even after mutating the conserved cysteine residue at its active site. They all say that its not because of autocatalytic property of the enzyme its because of some proteases from E.coli. Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
