With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Wed, 27 Jun 2012 10:14:58 -0400 >From: CCP4 bulletin board <[log in to unmask]> (on behalf of "R. M. Garavito" <[log in to unmask]>) >Subject: Re: [ccp4bb] The effect of His-tag location on crystallization >To: [log in to unmask] > > Most of the comments you will get will be anecdotal > in that people will report the successful results > and do not take the time or effort to characterize > the less successful results. This often occurs > because the tagged portion of the protein is most > often disordered, even in the best crystals. Thus, > other than saying "tagging on this end works, but > tagging on that end doesn't," there is little more > you can say. Each case will be different, and it is > almost impossible to arrive at any generalized > conclusion. > We prefer C-terminal tagged proteins for a number of > reasons, but if an N-terminally tagged protein > crystallizes well, so be it. Of the dozens of N- > and C-tagged protein structures we have solved in my > lab and with collaborators, I have only seen one > case of an ordered His-tag: the His residues had > coordinated Cd ions, which proved essential for > getting good crystals. However, beyond that there > was not much more to say. > For your protein and the resulting crystals, an > N-terminally tagged protein crystallized well. > Whether you can draw any more conclusions from > these results depends on characterizing crystals of > both N- and C-tagged proteins. Just assuming that > the C-tagged protein is trying to crystallize in the > same or related crystal form as the N-tagged protein > is an unwarranted assumption without experimental > evidence to back it up. That is why most groups > just run with the winner. > Cheers, > Michael > **************************************************************** > R. Michael Garavito, Ph.D. > Professor of Biochemistry & Molecular Biology > 603 Wilson Rd., Rm. 513 > Michigan State University > East Lansing, MI 48824-1319 > Office: (517) 355-9724 Lab: (517) 353-9125 > FAX: (517) 353-9334 > Email: [log in to unmask] > **************************************************************** > On Jun 26, 2012, at 9:06 PM, weliu wrote: > > Dear all, > > We crystallized a protein and found that crystal > quality greatly depended on the location of > His-tag. When a His-tag was added at the > C-terminus, only crystalline precipitate or > spherical quasi crystals were grown. However, when > the His-tag was moved to the N-terminus, single > crystals were grown under a number of conditions, > and the best one diffracted to 1.7 angstrom after > optimization. I was wondering if there were > published reports describing similar cases. > > Thank you in advance > > Wei Liu