Re: [ccp4bb] Problem with crystallization of Se-Met labeled protein
Consider also the possibility that your SeMet protein differs from the native protein by something other than the sulfur -- selenium. Growing your cells in different conditions may induce different host proteins that could modify your protein. (Often we use rich media for native protein production and minimal media of some flavor for SeMet) We had an example of this, (EJ Drake, Chem Biol, 13, 409) where growing the cells in minimal media (low iron, being the relevant factor) induced a host siderophore biosynthetic pathway that post-translationally added a cofactor to our recombinant protein. We only figured out why we couldn't crystallize the SeMet protein after we finally solved the structure (by a low homology molecular replacement) and saw that the site of cofactor addition was buried against a symmetry related molecule. Adding the 350Da cofactor at this position likely prevented the SeMet crystals from growing. Bottom line, if there is any chance of a post-translational modification, make sure your growth conditions are as similar to native protein expression as possible. A methionine auxotroph may be preferred over metabolic inhibition in this case. Andy On 5/27/08 5:11 PM, Joe Smith [EMAIL PROTECTED] wrote: Dear all, Sorry for an off-topic query. I have been unable to crystallize a Se-met containing protein (8 Met in 206 amino acids) in the native crystallization condition ( 0.1 M Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). As expected, solubility of Se-Met containing protein is little less than the wild type. Other than seeding, i don't know what else I should try for obtaining a Se-met crystal for phasing. Can I mutate some of the exposed Met (based on secondary structure prediction and homologous structure) to Ala as I feel I don't really need 8 Se for phasing 208 aa long polypeptide. I want to know what generally one should do when Se-Met containing proteins fail to crystallize. Thanks in advance. Joe PS: Since, protein contains 3 Cys residues.. I am also planning to try my luck with heavy atom compounds containing Hg. -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Senior Research Scientist Hauptman-Woodward Institute Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
[ccp4bb] Problem with crystallization of Se-Met labeled protein
Dear all, Sorry for an off-topic query. I have been unable to crystallize a Se-met containing protein (8 Met in 206 amino acids) in the native crystallization condition ( 0.1 M Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). As expected, solubility of Se-Met containing protein is little less than the wild type. Other than seeding, i don't know what else I should try for obtaining a Se-met crystal for phasing. Can I mutate some of the exposed Met (based on secondary structure prediction and homologous structure) to Ala as I feel I don't really need 8 Se for phasing 208 aa long polypeptide. I want to know what generally one should do when Se-Met containing proteins fail to crystallize. Thanks in advance. Joe PS: Since, protein contains 3 Cys residues.. I am also planning to try my luck with heavy atom compounds containing Hg.
Re: [ccp4bb] Problem with crystallization of Se-Met labeled protein
Have you thought about phasing off the sulfurs? This is quite a common technique nowadays. Jim On Tue, 27 May 2008, Joe Smith wrote: Dear all, Sorry for an off-topic query. I have been unable to crystallize a Se-met containing protein (8 Met in 206 amino acids) in the native crystallization condition ( 0.1 M Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). As expected, solubility of Se-Met containing protein is little less than the wild type. Other than seeding, i don't know what else I should try for obtaining a Se-met crystal for phasing. Can I mutate some of the exposed Met (based on secondary structure prediction and homologous structure) to Ala as I feel I don't really need 8 Se for phasing 208 aa long polypeptide. I want to know what generally one should do when Se-Met containing proteins fail to crystallize. Thanks in advance. Joe PS: Since, protein contains 3 Cys residues.. I am also planning to try my luck with heavy atom compounds containing Hg.
Re: [ccp4bb] Problem with crystallization of Se-Met labeled protein
Hi, Unless you've already tried exhaustively - why not to try MIR, as you mentioned - there are many derivatives out there to be tried and with a small protein the chances are pretty good. All you need is one good derivative :) This certainly sounds easier than mutating exposed Met! Cross-seeding with S-Met crystals into Se-Met protein worked for me on several occasions. Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Joe Smith Sent: Tuesday, May 27, 2008 5:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Problem with crystallization of Se-Met labeled protein Dear all, Sorry for an off-topic query. I have been unable to crystallize a Se-met containing protein (8 Met in 206 amino acids) in the native crystallization condition ( 0.1 M Tris pH 8.5, 1.2 M K-Na-tartrate; Theoretical pI of protein is 8.4). As expected, solubility of Se-Met containing protein is little less than the wild type. Other than seeding, i don't know what else I should try for obtaining a Se-met crystal for phasing. Can I mutate some of the exposed Met (based on secondary structure prediction and homologous structure) to Ala as I feel I don't really need 8 Se for phasing 208 aa long polypeptide. I want to know what generally one should do when Se-Met containing proteins fail to crystallize. Thanks in advance. Joe PS: Since, protein contains 3 Cys residues.. I am also planning to try my luck with heavy atom compounds containing Hg.
