Dear All,
We have a very similar problem, I have got a protein of 200 residues
predicted to have two free cys (but 14 internal cys!), 1 free histidine, 2
methionines (all predicted to be free).
Native crystals diffracted to 2.2 A was obtained in 200 mM KSCN, 20% PEG
3350, 13% glycerol, bis tris propane pH 7.5, 20mM spermine, 50mM
DTT(absolutely needed to obtain crystal). We tried iodide soak and sulphur
SAD but phasing was not promising.
From all the posts I have read, mercury seems to be the best to try to
derivatise it but probably needs to remove DTT.
My Question is:
Does mercury tends to get into the protein core to denature protein or not?
For gel-shift assay, do people normally use a special gel tank for heavy
metal work?
thanks in advance
Xiaoli Xiong (Alex)
--On 15 July 2009 14:33 +0200 Sebastiano Pasqualato
<sebastiano.pasqual...@ifom-ieo-campus.it> wrote:
Hi all,
I've got crystals of a protein of ca 200 residues, with 2 free
cysteines, 5 histidines, 2 methionines.
We have nice diffraction for the native crystals, that grow in 150 mM
KSCN, 17% PEG 3350, bis tris propane pH 8.8.
We are crystallising the SeMet derivative, but I'm not completely sure I
will be able to have nice crystals by Saturday, when we have tunable
time at the ESRF.
I was thinking of trying with some heavy atom soaks, but only have like
30 crystals, so limited trials allowed!
Which compound would you advice as more likely to work, and thus worth
testing?
Thanks in advance for the suggestions,
ciao
s
--
Sebastiano Pasqualato, PhD
IFOM-IEO Campus
Dipartimento di Oncologia Sperimentale
Istituto Europeo di Oncologia
via Adamello, 16
20139 - Milano
Italy
tel +39 02 9437 5094
----------------------
Xiaoli Xiong
PhD Candidate
Department of Cellular and Molecular Medicine
School of Medical Sciences
University of Bristol
University Walk
Bristol BS8 1TD, UK
x.l.xi...@bristol.ac.uk
