Hi Klaus,
- small molecular weight PEG's e.g. 200 instead of DMSO, has the advantage of
also helping to cryo protect
- Methanol (only for dispensing the compound into wells) then allow to
evaporate and simply add your cryo-protected crystals, the hope is that
sufficient of your ligand goes into solution
- different method but also efficient, use your protein as "solubilizer" .
Figure out the lowest concentration of DMSO under which your ligand is soluble
(maybe 0.5%) in solution at say 50 µM then take your diluted protein and mix it
with the ligand, then concentrate to the required concentration needed for
crystallization
Jürgen
On May 23, 2013, at 9:36 AM, Klaus Fütterer wrote:
Dear CCP4BB followers,
We are currently trying to obtain ligand-bound complexes for one of our
proteins by soaking and/or co-crystallisation. We have had prior success for
this protein, but using a different class of ligands. The new ligand (in DMSO)
remains in solution (more or less) when mixed with the
reservoir/cryoprotectant, and the diffraction pattern survives the soaking
nicely. Annoyingly though, all we see are density peaks that match the size of
DMSO and become more pronounced when increasing [DMSO] (to help solubilisation
of the ligand). Soaking times varied between minutes to 16 hours. Kd was
measured ( ~ 10 uM) in the solution state.
We have tried pyridine (which keeps the ligand in solution, but kills
diffraction in an instant), and DMF (which doesn't keep the ligand in solution
when mixing with cryoprotectant).
I am wondering whether the community has suggestions for alternative organic
solvents that have been used to solubilise hydrophobic ligands, and are
reasonably gentle to the protein crystal.
Thank you.
Klaus
=======================================================================
Klaus Fütterer, Ph.D.
Reader in Structural Biology
Undergraduate Admissions
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E:
k.futte...@bham.ac.uk<mailto:k.futte...@bham.ac.uk>
Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab
=======================================================================
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://lupo.jhsph.edu
