Hi vito,
I don't think you need double labelling. Two (if there is a Met at the N-term
it might be less ordered) or three SeMet should suffice to phase 360 residues.
It was true in the old days that a Se could phase less than a hundred residues,
but if your measurement is good (resolution
I have only heard of a few cases of successful Se incorporation into
Cys, and none of them sounded like fun. Sounds like you have gotten a
few suggestions already. There is not much literature on this.
As for alternative solutions to your original question, I can tell you
the success or
Dear Vito
AMPLE is another option to easily and automatically find conserved cores
among a set of distant homologues. You point it to a directory
containing your homologues and use the
-homologs True
flag. You can use the command line or CCP4i. It will then use GESAMT to
find the multiple
Yes, I agree that MR is worth a shot, though depending on the resolution your
life may be vastly easier with experimental phases! We've had several cases
where the following kind of procedure works: find related structures with a
very sensitive homology search (we like HHPRED, though other
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, yo
09 am
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling
I second (or third) the suggestion that others have given to try soaking
mercury compounds into your crystal. Mercury absolutely loves free cysteines,
and if you have 5, you have a great chance of
I second (or third) the suggestion that others have given to try soaking
mercury compounds into your crystal. Mercury absolutely loves free cysteines,
and if you have 5, you have a great chance of getting binding. If isomorphism
is maintained after soaking, the isomorphous differences will be
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
Dear Vito,
for SeMet have a look here:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins
Worked like a charm for E.coli and also for other expression hosts
with minor modifications
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
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
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