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 bette
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 fai
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 s
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 opti
l
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 o
L.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 getting binding. If isomorphism
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
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
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
(https://www.nature.com/nature/journal/v516/n7529/full/nature14003
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@JISC
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 protocol
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 ex
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