Re: [ccp4bb] Se-Met and disulfides
Hi Reza, You have here an example of MAD data collected fromoxidized and reducedselenomethionine-containing protein: It states :"phasing power of theoxidized data is doubled for the dispersive signal and is 20% stronger for theanomalous signal at the peak wavelength" Thomazeau K, Curien G, Thompson A, Dumas R, Biou V. MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Crystallogr D Biol Crystallogr. 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 23. PubMed PMID: 11526338. Daniel Le 27/02/2014 17:05, Reza Khayat a écrit : Hi, I'm sure this question has been addressed before and I apologize for asking it again. I'm working on a protein predicted to have multiple disulfide bonds. The protein is expressed in an E. coli strain with an "oxidizing" cytoplasm, and is purified under nonreducing conditions (no DTT, b- ME...). I'd like to produce some Se-Met incorporated protein for phasing. Given that Se-Met can oxidize, its anomalous scattering is dependent on its oxidation state, and a mixture of oxidized and reduced Se-Met makes phasing difficult, what is the suggested protocol to follow in my situation? Here's hoping that the solution is not dumping a bunch of Se-Met into all the purification buffers... Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message Date: Thu, 27 Feb 2014 11:22:10 + From: CCP4 bulletin board (on behalf of Vicky Tsirkoni ) Subject: [ccp4bb] To: CCP4BB@JISCMAIL.AC.UK Hi Prerana, You could try 4°C as well (dont forget to use lower protein concentration) in my case this worked better. Cheers, Vicky G. Tsirkone, MSc Laboratory for Biocrystallography Department of Pharmaceutical and Pharmacological Sciences KU Leuven O&N II Herestraat 49 - box 822 3000 Leuven | Belgium Tel.: +32 16 3 23419 e-mail: vicky.tsirk...@pharm.kuleuven.be From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. [tracy...@gmail.com] Sent: Monday, February 24, 2014 5:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Hi Vicky, I tried to change the ratio of paraffin:silicon oil to 1:2, but still the protein precipitated. So I tried to do it other way round, I kept paraffin:silicon oil ratio as 2:1 and so far it has not precipitated. Apart from that, I separately used the two oils.In silicon oil there was immediate precipitation, but none in parafin oil. Prerana
Re: [ccp4bb] Se-Met and disulfides
I wouldn't worry about oxidation of SeMet; at least I never have. A number of years back there was a paper published actually claiming that oxidised SeMet has a higher and sharper absorption edge than reduced SeMet. Of course mixed forms could complicate things but I would just purify like you do for the WT protein. Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Thursday, February 27, 2014 4:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Se-Met and disulfides Hi, I'm sure this question has been addressed before and I apologize for asking it again. I'm working on a protein predicted to have multiple disulfide bonds. The protein is expressed in an E. coli strain with an "oxidizing" cytoplasm, and is purified under nonreducing conditions (no DTT, b- ME...). I'd like to produce some Se-Met incorporated protein for phasing. Given that Se-Met can oxidize, its anomalous scattering is dependent on its oxidation state, and a mixture of oxidized and reduced Se-Met makes phasing difficult, what is the suggested protocol to follow in my situation? Here's hoping that the solution is not dumping a bunch of Se-Met into all the purification buffers... Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message >Date: Thu, 27 Feb 2014 11:22:10 + >From: CCP4 bulletin board (on behalf of Vicky Tsirkoni ) >Subject: [ccp4bb] >To: CCP4BB@JISCMAIL.AC.UK > > Hi Prerana, > You could try 4°C as well (dont forget to use > lower protein concentration) in my case this worked > better. > Cheers, > Vicky G. Tsirkone, MSc > Laboratory for Biocrystallography > Department of Pharmaceutical and Pharmacological > Sciences > KU Leuven > O&N II Herestraat 49 - box 822 > 3000 Leuven | Belgium > Tel.: +32 16 3 23419 > e-mail: vicky.tsirk...@pharm.kuleuven.be > > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on > behalf of Prerana G. [tracy...@gmail.com] > Sent: Monday, February 24, 2014 5:23 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] > Hi Vicky, > I tried to change the ratio of paraffin:silicon oil > to 1:2, but still the protein precipitated. So I > tried to do it other way round, I kept > paraffin:silicon oil ratio as 2:1 and so far it has > not precipitated. Apart from that, I separately used > the two oils.In silicon oil there was immediate > precipitation, but none in parafin oil. > Prerana
[ccp4bb] Se-Met and disulfides
Hi, I'm sure this question has been addressed before and I apologize for asking it again. I'm working on a protein predicted to have multiple disulfide bonds. The protein is expressed in an E. coli strain with an "oxidizing" cytoplasm, and is purified under nonreducing conditions (no DTT, b- ME...). I'd like to produce some Se-Met incorporated protein for phasing. Given that Se-Met can oxidize, its anomalous scattering is dependent on its oxidation state, and a mixture of oxidized and reduced Se-Met makes phasing difficult, what is the suggested protocol to follow in my situation? Here's hoping that the solution is not dumping a bunch of Se-Met into all the purification buffers... Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message >Date: Thu, 27 Feb 2014 11:22:10 + >From: CCP4 bulletin board (on behalf of Vicky Tsirkoni ) >Subject: [ccp4bb] >To: CCP4BB@JISCMAIL.AC.UK > > Hi Prerana, > You could try 4°C as well (dont forget to use > lower protein concentration) in my case this worked > better. > Cheers, > Vicky G. Tsirkone, MSc > Laboratory for Biocrystallography > Department of Pharmaceutical and Pharmacological > Sciences > KU Leuven > O&N II Herestraat 49 - box 822 > 3000 Leuven | Belgium > Tel.: +32 16 3 23419 > e-mail: vicky.tsirk...@pharm.kuleuven.be > > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on > behalf of Prerana G. [tracy...@gmail.com] > Sent: Monday, February 24, 2014 5:23 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] > Hi Vicky, > I tried to change the ratio of paraffin:silicon oil > to 1:2, but still the protein precipitated. So I > tried to do it other way round, I kept > paraffin:silicon oil ratio as 2:1 and so far it has > not precipitated. Apart from that, I separately used > the two oils.In silicon oil there was immediate > precipitation, but none in parafin oil. > Prerana
