Thanks everyone for the pointers. I should clarify - my issue here is the
multiple stage "disconnect" between PDB in xtal1 -> cutout density (into a
"interim cell" just for this stage) -> phaser MR with density search model into
xtal2
Hence, do I just simply pop the model from (1) into the "int
Dear All,
I suspect the way out of this is a new crystal (!) but interested to hear any
advice.
I have two crystal forms of a 500aa protein, vaguely tube-shaped
1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map indicates it
is a relative of other folds (but those are not so clos
agree, any crystallisation idea is worth pursuing, given you have or can make
enough sample to try it with.
having said that, wouldn't you tend to select for the same crystals as the
seed, i.e. crystals of the component on its own?
have you tried limited proteolysis of your sample, incl. a bit of
Hi Peter
This idea was discussed at the recent RAMC meeting, and there is at least
one example where it has worked.
Generally, cross-seeding can work as long as you have homology. See e.g.
Obmolova et al. Acta Crystallogr. 2010, D66, 927–933. The same group has
reported seeding a complex with
agree, any crystallisation idea is worth pursuing, given you have or can make
enough sample to try it with.
having said that, wouldn't you tend to select for the same crystals as the
seed, i.e. crystals of the component on its own?
have you tried limited proteolysis of your sample, incl. a bit
The idea is not at all crazy. In a sense it is quite similar to
Stoichiometric variation screening* if you consider that the lattice of
the crystallized subunit may contain planes
that might be conserved in the crystal of your hope for 3 protein complex.
*Stura, E.A., Graille, M., Taussig, M
I forgot to mention, I can reconstitute the complex (co-expression/mixing
proteins) and the complex comes off both ion exchange and an SD200 as one peak.
Just haven't had luck with getting crystals.
On Wed, 2011-09-21 at 18:04 +0100, Peter Hsu wrote:
> Or is this just a crazy/bad idea?
If there is one thing that I learned about crystallization, is that very
few ideas are so crazy that they are bad (i.e. not worth trying). Well,
if dried seaweed and ground horse hair are good for seeding, I d
Hi all,
I've been trying to crystallize a 3 protein complex recently with little
success. However, crystals of each subunit have previously been crystallized. I
was wondering if any one knows of any literature/experiences where people have
used seeds from an individual subunit to seed for a com
For gel exclusion chromatography it is generally adsvisable to include
100 mM NaCl or equivalent ionic strength in the elution buffer to
suppress nonspecific adsorption. In addition the pH and ionic strength
of the elution buffer should be compatible with your protein folding
stability. It is possi
Hello
I am trying to complex two proteins of 57 and 18 kda and using pET 22b as an
expression system. I purified both with His tag and the purity is quite nice
after Ni-NTA (Buffer is Tris,Nacl and Imidazole). After purification with
Ni_NTA i tried to improve my protein purity using gel
filteration
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Office 2004 Test Drive User
Sent: Thursday, May 21, 2009 3:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Complex degradation
Dear members,
I am trying to crystallize a complex of two proteins
Dear members,
I am trying to crystallize a complex of two proteins prepared in Tris, NaCl
and DTT. The problem is one of the proteins degrades over time and allows
only the second protein to crystallize. While addition of protease
inhibitors such as EDTA is one of the ways to go, I would like to kn
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