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 +0000
From: CCP4 bulletin board <[email protected]> (on behalf
of Vicky Tsirkoni <[email protected]>)
Subject: [ccp4bb]
To: [email protected]
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: [email protected]
----------------------------------------------------
From: CCP4 bulletin board [[email protected]] on
behalf of Prerana G. [[email protected]]
Sent: Monday, February 24, 2014 5:23 PM
To: [email protected]
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
