We have successfully used non-auxotrophic strains for incorporation of SeMet and SeCys, with an incorporation level of >80% and no effects on yield or solubility. Details can be found here:
http://scripts.iucr.org/cgi-bin/paper?S0907444910042022 It's a straight-forward protocol and structure solution was trivial, even without 100% incorporation. With your number of Met and Cys, it should give you enough signal, as long as expression is not affected by minimal media. You can do a small test just to ensure protein expression is OK before doing large scale purification. Using derivatives is another option but it might be time consuming and extensive to search for appropriate heavy atoms, so this might be good alternative. Good luck! Paula =================================== Dr Paula S. Salgado Lecturer in Macromolecular Crystallography Institute for Cell and Molecular Biosciences Faculty of Medical Sciences 3rd Floor Cookson Building Newcastle University Newcastle upon Tyne, NE2 4HH, UK Tel: +44 (0)191 208 7432 Fax: +44 (0)191 208 7424 Email: paula.salg...@ncl.ac.uk ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Bonsor, Daniel <dbon...@som.umaryland.edu> Sent: 21 June 2017 22:03:06 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling To perform double labelling on your protein in an NON auxotrophic strain may be difficult. For the seleomet side, you can shut down the biosynthesis of Met by the addition of lysine, phenylalanine, threonine, isoleucine and valine; various protocols exist online. However to shutdown biosynthesis of cysteine, you need to add cysteine (acts as a negative feedback inhibitor on itself ), which defeats the point. I cannot find online if selenocys can inhibit biosynthesis of cysteine, like cysteine can. You could perform a small test expression by adding all the amino acids (lysine, phenylalanine, threonine, isoleucine, valine, selenomet, selenocys) 30 mins before induction, do your typical expression and purification and confirm by mass spec of labelling. If not you could trying sulfur SAD, the various derivatives suggested today, soaks with halides, magic triangle. Mutate leucine residues to methionine. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Vito Calderone Sent: Wednesday, June 21, 2017 11:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Se-Met and Se-Cys double labelling I am working on a protein having 360 residues. In its sequence there are 3 Met and 5 free Cys. I will need MAD to solve the structure since based on the sequence the closest homologue has 20% identity...I suppose MR would be very unlikely to work...so I would like to express a selenium derivative to exploit MAD. Looking in the literature 1 Se-Met every 120 residues seems not to comply the threshold to get a good anomalous signal. For this reason I would like to exploit both Met and Cys so I would have 8 seleniums per 360 residues. Could somenone suggest a reference to a protocol to express the double mutant protein in NON auxotrophic strains of E. coli which you have experienced working efficiently? Thanks
