Something like Balbes for MR is worth a shot? I wouldnt bother manually with MR anymore unless really clear. Or try Hg and Pt for Cys, His and Met. I would try the Pt chlorides. All depends on pH etc...
Tommi -------- Alkuperäinen viesti -------- Lähettäjä: "Keller, Jacob" <kell...@janelia.hhmi.org> Päivämäärä: 21.06.2017 19.51 (GMT+02:00) Saaja: CCP4BB@JISCMAIL.AC.UK Aihe: Re: [ccp4bb] Se-Met and Se-Cys double labelling Halide soaks anyone? Cs or NaI? Jacob From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Wednesday, June 21, 2017 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling If your data is good enough, your SeMets alone might well be enough. Soaking native crystals in Hg compounds may also work, avoiding SeMet altogether. We have had a lot of success with methylmercury chloride binding to free Cys. You may have to experiment with different soaking times and protocols. Finally, don't give up on MR too early, what matters is the structural similarity, not directly the sequence identity. We've had success once with 19% identity. Native protein may be much easier to produce than the SeMet and SeMet/SeCys versions (and may differ a lot between proteins). Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij On 21 Jun 2017, at 17:46, Vito Calderone <calder...@cerm.unifi.it<mailto:calder...@cerm.unifi.it>> wrote: 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匢 suppose MR would be very unlikely to work卻o 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
