Additional to all other suggestions you can also mix your ligand at
low concentration to lots of low concentrated protein and then
concentrate your protein over time. The advantage, ligands with low
solubility can bind to your protein while you are concentrating it.
And then I would attempt to set up trays with your known
crystallization conditions and eventually cross-seed the drops with
non-ligand crystals.
Good luck,
Jürgen
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
On Nov 13, 2009, at 3:35, "herman.schreu...@sanofi-aventis.com" <herman.schreu...@sanofi-aventis.com
> wrote:
Dear Rongjin,
In addition to what the others said, I would do the following:
1) check your apo-structure to make sure the putative ligand binding
is not blocked by a crystal contact and also check that there is a
channel leading to the binding site. You may not get hits in your
cocrystallization experiments because the ligands interfere with a
crystal contact. If the binding site is blocked, you need a
completely different crystal form. Best would then be to do a de
novo screening in the presence of the most potent ligand.
2) get independent evidence that your ligand does bind. This can be
an (enzyme) assay, NMR, biacore, ITC etc. If you are lucky, you may
be able to also check whether compounds of your crystallization
buffer or DMSO are interfering with binding.
3) Take the most soluble and most potent compound from (2) and soak
in the highest concentration you can achieve, which may be as high
as 0.5-1.0 M. First try to dissolve the compound in water and only
if that does not work, try ethanol or DMSO.
4) Try to soak crystals grown under very different conditions. We
recently had a case where we could not soak-in compounds in the
presence of PEG, but high-salt was no problem. I know cases where
the reverse was true, where high salt was preventing binding.
Good luck!
Herman
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf
Of Rongjin Guan
Sent: Thursday, November 12, 2009 10:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] small molecule soaking screening
Hi All
Sorry that this is a non-ccp4 question, but I hope I can get some good
suggestions from the community.
We have protein crystals under various conditons and want to soak them
with different potential inhibitors. Most of inhibitors have very
small
molecular weights (200-300), so it become a problem how to detect if
the
small compounds have been soaked into the crystals or not.
(co-crystallization experiments yielded no hits so far, though the
free
form is easy to be crysatllized under many conditions)
We pay $500/day for local X-ray facility access, so we wonder if
there are
some more efficient ways that allow us to know if the small compounds
soaked in or not, without collecting a whole data set for MR.
We are also thinking if we can mix several compounds together for
soaking,
to reduce the combinations of soaking experiments with various
compounds
and crystals from various conditions. Is this practical, if some of
them have
pretty similar binding affinities to the protein?
All comments/suggestions are welcome.
Thank you
Rongjin Guan
