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Yangming, This topic has been discussed before - basically there are no easily discernable trends - some proteins crystallize better with a tag, others - without, and yet others - don't care whether the tag is there or not. I tend to try either, just to see what works better (time and effort permitting). C-terminal tags are messier to remove because most known proteases require considerable P(-X) segments. There are at least two clever ways to cleave C-terminal His-tags 'cleanly' but they're generally not worth the trouble, IMHO. N-terminal tags are easy to cleave since several 'clean' site-specific proteases are available (TEV, TVMV, thrombin, etc.). I am not sure what exactly do you mean by your second question - however there are cases when even one amino acid difference results in huge changes in crystallization behavior. The effect greatly depends on the location of the change - if your proposed tag location is on or near a crystal contact then the change will likely be huge. Then again, there are (we think) clever ways to play with protein surfaces to elicit crystallization behavior - case in point is our recent structure where three amino acid changes resulted in huge improvement of crystals for a 2300 amino acid heterotetrameric protein. Morale of this shameless self-promotion is that small changes of protein surface can have profound effects on crystallization, and since tags are on the surface the effects may range from none to huge. Happy protein engineering, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Yanming Zhang Sent: Monday, February 04, 2008 8:54 PM To: [log in to unmask] Subject: [ccp4bb] His tag Hi All, Maybe, I should not have asked this question: Can anybody give me the hints (or point to the references) on the impact of His tag on crystallization experiments. In perticular: 1, With or without His tag, which one is better for crystallization? 2, If I successfully crystallized N-terminal, what information can I get to assist my set-up of C-terminal crystallization set-up. Sorry for taking up your time because of my inadequate knowledge Thank you for your help Yanming