As said, your Km is different in mother liquor than in your reaction conditions, but even that is not the end of it: You ligand/substrate might be inducing the slightest of all conformations in your protein that interferes with crystallization, or might block crystal contacts. Then, you can either (1) get no crystals any longer, or (2) get no substrate in your crystals, depending on whichever wins energetically, substrate binding or crystal formation. Therefore, being orders of magnitude above physiological Km/Kd is still not a guarantee for getting crystals of a complex. So, dwelling obsessively on molar ratios and scratching your head becomes irrelevant after a while, one should just do the experiment with a good molar ratio as suggested in this thread, and accept the verdict of the gods of crystallization.
Interestingly enough, your substrate-protein complex might crystallize more efficiently, or crystallize in different conditions, so not all is negative. Engin P.S. You are talking about Km and a substrate, but I hope you have a substrate analog, inactive enzyme or you are trying to capture the product complex, otherwise you might be in for a surprise when you solve the structure. ----- Original Message ----- From: "Juergen Bosch" <[EMAIL PROTECTED]> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, December 1, 2008 6:35:24 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] co-crystallization The Km changes with your reservoir, so predictions are limited. In general if you have a low Km this is favourable but not a given that your ligand will be found in the electron density map. As a starting point try a molar ratio of >3 of the ligand to your protein and you can go as high as you want (assuming it's soluble and does not cost you an arm and a leg). 50 fold excess has been used in some cases. And remember for your cryo-step to include your ligand in sufficient amounts ! Jürgen On 1 Dec 2008, at 05:31, yangliuqing wrote: Hello,everyone, I have a question for cocrystallization, is there some relationship between Km value and substrate concentration when making cocrystallization? How can I know the substrate is enough for binding? Thank you very much! liuqing 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
