Hi Xiaopeng,
In addition to what has been suggested:
Make sure to understand that your 'inhibitors' are really inhibitors and
not just general destabilizers of proteins. A binding assay like ITC or
Thermofluor like assay is useful to establish that your inhibitors are
actually binding to the protein and not just unfolding it. Destabilizers
tend not to show up in crystal structures.
Accessibility of the active site is also key to success. Check for any
intrusions of symmetry mates into the putative binding pocket. This is a
common problem for proteases for instance. If so, you would need to
screen for a different apo-crystal form or try co-crystallization.
Try soaking in cryo-protectant with excess ligand present for a couple
of days. Solubilizing additives like 3% DMSO or cyclodextrane may also
help. If your protein crystallizes in high salt, you can try to switch
soaking in higher PEG concentrations, that sometimes improves things.
Good luck
Carsten
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of
> ???
> Sent: Saturday, August 28, 2010 1:40 AM
> To: [email protected]
> Subject: [ccp4bb] Problem with getting protein-inhibitor complex
> structure
>
> Dear all,
> We are working on protein-inhibitor complex crystal structures,
> although we did get nice crystals and soaked them with inhibitor for
> several days, all structures we obtained were empty!!There are nothing
> at the active site. For several crystals. their color turned very
> clearly from colorless to yellow the color of the inhibitors, nothing
> found either.
> These inhibitors work very well in enzyme assay. We, especially
our
> students, are very depressed...
> Any suggestions will be really appreciated!
>
>
>
>
> Xiaopeng Hu, D.Sc.,
> School of Pharmaceutical Sciences
> Sun Yat-sen University,
> Higher Education Mega Center
> Guangzhou, Guangdong, China 510006
> Tel. +86 020 39943032
