Dear Fritz,
You are right. The recollection of that striking 2015 paper was somehow
too strong to be 100% faithful: these are two different modes of using the
ligand deposited on a solid support by drying a solution of it, to avoid the
DMSO problem.
Best wishes,
Gerard.
--
On Fri, Oct 04, 2024 at 10:17:45PM +0200, Guenter Fritz wrote:
> :)
> Gerard,
> "soaking"!
> Thanks for the reference!
> cheers
> guenter
> >Dear Guenter,
> >
> > The first description of the idea behind this technique that I am aware
> >of is that published almost 10 years ago by a French group under the name of
> >"'dry' co-crystallisation" in the following paper:
> >
> > http://dx.doi.org/10.1107/S1399004715010342
> >
> >
> > Best wishes,
> >
> > Gerard
> >
> >--
> >On Fri, Oct 04, 2024 at 05:40:12PM +0200, Guenter Fritz wrote:
> >>Hi Amit,
> >>like Artem wrote below, even despite poor solubility, soaks with
> >>dried compounds work well (also in our hands).
> >>We adapted it from the work of T. Barthel, J. Wollenhaupt and
> >>Manfred Weiss. Also colleagues from industry report good results.
> >>We add compound dissolved in DMSO to the crystallization well, let
> >>DMSO evaporate overnight (37° incubator), then put mother liquor on
> >>top of the dried compound and then the crystals. If all compound
> >>would dissolve again, final concentration would be 10-50 mM. Soak
> >>time from 1-2 h to overnight.
> >>This worked for poorly soluble compounds where we did not succeed
> >>with soaking in the presence of DMSO.
> >>Hope that helps!
> >>Best,
> >>Guenter
> >>
> >>>Dear Amit
> >>>
> >>>As David already pointed out, all proteins are different and it's
> >>>hard to say in advance what amount of DMSO may work (or not).
> >>>
> >>>An additional concern is that DMSO can also interfere with ligand
> >>>binding (cases from my personal past history), especially if these
> >>>inhibitors/ligands are on the weaker side.
> >>>
> >>>Solutions:
> >>>
> >>>Despite its very high boiling point (189C) DMSO can in fact be
> >>>evaporated from a small sample of your inhibitor, resulting in
> >>>more or less solid inhibitor sample that can be re-dissolved in
> >>>the same DMSO (but higher concentration), some other solvent, or
> >>>perhaps directly in the protein solution. The latter is sometimes
> >>>the only way to do this - I used to set up drops of DMSO
> >>>solutions, then evaporate the DMSO in high vacuum (heating helps)
> >>>with a cryofinger, then set up protein drops on top. This of
> >>>course requires access to a lyophilizer or something similar.
> >>>
> >>>If you have a vial of your solution you can freeze-dry DMSO with
> >>>water, by first diluting the sample then freeze-drying it. Also
> >>>water can sometimes crash the substance out (if not water, then
> >>>perhaps Ether or another solvent where your inhibitor does not
> >>>dissolve) which makes it easier to redissolve (but there will be a
> >>>loss of course).
> >>>
> >>>Find a friendly chemist nearby and ask then to put your sample in
> >>>a speedvac on 'high BP' setting
> >>>
> >>>Notably, if you're "blessed" with an inhibitor that has the
> >>>general solubility of a Sony Walkman, once you get rid of the
> >>>DMSO, you may find out that the damned thing does not want to
> >>>dissolve in anything else, including your protein solution. This
> >>>happens a lot during early discovery phases when compounds are not
> >>>very active (micromolar) and also poorly soluble (also
> >>>micromolar). This is by far the most frequent cause for failing to
> >>>co-crystallize (or soak) a ligand of interest. Very frustrating.
> >>>Some success can be achieved using high DMSO or DMF (DMA also can
> >>>be good) in your crystallization, or by phase transfer catalysts
> >>>like Cyclodextrin(s) or appropriately formulated micelles. All of
> >>>which can also mess up crystallization, needless to say.
> >>>
> >>>Best of luck in your endeavors!
> >>>
> >>>Artem
> >>>
> >>>- Cosmic Cats approve of this message
> >>>
> >>>
> >>>On Wed, Oct 2, 2024 at 2:51 PM amit gaur <cdriamitg...@gmail.com> wrote:
> >>>
> >>> Hi everyone,
> >>>
> >>> I am trying to crystallize a protein with a drug molecule. The
> >>> protein concentration is 15.5 mg/ml, the drug stock concentration
> >>> is 10 mM, and the drug is dissolved in DMSO. I am adding the drug
> >>> to a final concentration of 1 mM in 100 ul of protein, and the
> >>> DMSO volume is 10 ul for Co-crystallization. I want to know how
> >>> much DMSO is permissible during co-crystallization with the drug
> >>> and if DMSO can poison crystal formation. I have not been
> >>> successful in getting crystals with inhibitors till now, but I
> >>> obtained crystals of protein without DMSO, and those diffracted to
> >>> 2.5A.
> >>>
> >>> Thanks,
> >>>
> >>> *Dr. Amit Gaur,*
> >>> *Research Scientist*
> >>> *Center for Biotechnology and **Interdisciplinary Studies,*
> >>> *Rensselaer Polytechnic Institute,*
> >>> *1623 15th Street, Troy, NY, 12180*
> >>>
> >>> *
> >>> *
> >>>
> >>>
> >>> ------------------------------------------------------------------------
> >>>
> >>> To unsubscribe from the CCP4BB list, click the following link:
> >>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> >>> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1>
> >>>
> >>>
> >>>------------------------------------------------------------------------
> >>>
> >>>To unsubscribe from the CCP4BB list, click the following link:
> >>>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> >>><https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1>
> >>>
> >>########################################################################
> >>
> >>To unsubscribe from the CCP4BB list, click the following link:
> >>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> >>
> >>This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing
> >>list hosted by www.jiscmail.ac.uk, terms & conditions are available at
> >>https://www.jiscmail.ac.uk/policyandsecurity/
########################################################################
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list
hosted by www.jiscmail.ac.uk, terms & conditions are available at
https://www.jiscmail.ac.uk/policyandsecurity/
