If the solubility is a problem, you can start out with a low concentrated but soluble solution of your inhibitor in a diluted protein sample. Then concentrate up your protein to the desired amount in the presence of the inhibitor, set up trays and be happy. Additionally you could add more inhibitor into your precipitant solution when setting up the trays. Since you are not screening for new conditions you could prepare a mixture of [protein-inhibitor]+inhibitor+reservoir solution and directly dispense this for crystallization over non-inhibitor containing reservoir solution.
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 Aug 28, 2010, at 8:16 AM, 胡小鹏 wrote: > Some inhibitors have bad solubility but low Ki (less then 1 uM), we keep > inhibitors solid in the soaking drops and don't know the exact concentration. > For inhibitors with good solubility, we keep the ration at 10 or higher, > unfortunately, Ki of these inhibitors are not good, for example, around 30 uM. > > > ----- 原始邮件 ----- > 发件人: "Jürgen Bosch" <[email protected]> > 收件人: "胡小鹏" <[email protected]> > 抄送: [email protected] > 发送时间: 星期六, 2010年 8 月 28日 下午 8:00:46 > 主题: Re: [ccp4bb] Problem with getting protein-inhibitor complex structure > > What concentration/ratio of your inhibitor did you use ? > > > Also check this out: (short soaks, see time course experiment) > Bosch et al. Using fragment cocktail crystallography to assist inhibitor > design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med > Chem (2006) vol. 49 (20) pp. 5939-46 > > > > 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 Aug 28, 2010, at 1:40 AM, 胡小鹏 wrote: > > > > 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 > > -- > -- > 胡小鹏 理学博士 > 教授 > 中山大学药学院结构生物学实验室 > 广东省 广州市 番禺区 大学城 510006 > Tel: 020-39943032 > > > Xiaopeng Hu, D.Sc., > Professor > School of Pharmaceutical Sciences > Sun Yat-sen University, > Higher Education Mega Center > Guangzhou, Guangdong, China 510006 > Tel. +86 020 39943032
