Masaki,
If your crystals crack when you add calcium it implies that calcium
binding induces a conformational
change.
You should try co-crystallization with an "epitaxial jump" approach:
Stura, E. A., Charbonnier, J.-B. & Taussig, M. J.(1999) Epitaxial jumps.
J. Cryst. Growth 196: 250-260.
Since it is likely that calcium binding destroys only one crystal contact,
you should screen for co-crystals
with seeds of your calcium-free crystals. One of the planes of your
calcium-free form should be able to stimulate
growth of crystals with calcium but your crystallization conditions are
likely to be different.
Without seeding you are likely to be hindered by the nucleation barrier
which seeding overcomes.
Enrico.
On Thu, 20 Feb 2014 11:29:47 +0100, Masaki UNNO <unn...@mx.ibaraki.ac.jp>
wrote:
Dear all
Apologies for the off-topic question:
We are studying an enzyme that is activated by Ca2+. We obtained the
crystals of the substrate and Ca2+-free form and solved the structure at
2.7
A resolution. However, the active site electron density map was not
clear,
although other regions are clear. We would like to determine the
substrate-complex with Ca2+, which will elucidate the active site
structure
at a higher resolution.
Now we have a problem that the crystals of a mutant which can bind the
substrate and Ca2+ always have cracks in soaking to the crystallization
solution containing CaCl2. Co-crystallization does not work at this
time. We
estimate the structural change in Ca2+-binding is not so big because the
isozyme structure did not change very much when binding Ca2+. An isozyme
structure in complex with the substrate was determined by soaking Ca2+
(and
the substrate).
How should we overcome this problem?
Best regards
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Masaki UNNO
Graduate School of Science and Engineering, Ibaraki University, Japan
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
