[ccp4bb] How to crystallize a helicase
Dear all! We've been trying to crystallize a helicase. No protein crystal was found after some typical conditions (kit I, kit II, and Index) were repeatly screened for more than three times. 50 mM MES (pH 6.0), 5% glycerol, and 1mM DTT is used as the final storage buffer. The most significant problem of this protein is its precipitation in most of the screening conditions. Protein concentration gradient was considered, but it can not work. I was wondering if there is any way to work aroud this problem. Look forward to your suggestions! Thanks! Art
Re: [ccp4bb] How to crystallize a helicase
Dear Art, It seems twisted, indeed. First of all you need to verify that your protein is at all in a good state (you seem to imply that it is not with your remark on protein concentration). Basic biophyscial characterisation for stability and structural homogeniety is required to reach proper buffer conditions for your protein, and you may consider a complex with relevant ligands Poul Dear all! We've been trying to crystallize a helicase. No protein crystal was found after some typical conditions (kit I, kit II, and Index) were repeatly screened for more than three times. 50 mM MES (pH 6.0), 5% glycerol, and 1mM DTT is used as the final storage buffer. The most significant problem of this protein is its precipitation in most of the screening conditions. Protein concentration gradient was considered, but it can not work. I was wondering if there is any way to work aroud this problem. Look forward to your suggestions! Thanks! Art
Re: [ccp4bb] How to crystallize a helicase
I would certainly try cocrystallizing with adp, amppnp, or ATPgS Many helicases have been crystallized in this way. Papers by Sawaya et al and Singleton et al come readily to mind. Best of luck Savvas On 09 Mar 2011, at 15:52, Ed Pozharski epozh...@umaryland.edu wrote: On Wed, 2011-03-09 at 10:52 +, Art wrote: No protein crystal was found after some typical conditions (kit I, kit II, and Index) were repeatly screened for more than three times. See Einstein's definition of insanity :) Protein concentration gradient was considered, but it can not work. Not sure what you mean by this, but you can also try diluting reservoir solutions. Also, given your protein buffer, you may be more or less locking yourself into a narrow pH range - try 10mM MES instead. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
