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You mght be able to force a compex in crystallization by attaching a linker between the two proteins. Then, they will have to be near each other, and the crystallization driving force will do the rest. Its been done before, but linker length can be tricky for if expression is affected so you may have to make a few constructs. Another possibility is to screen for a mutation/mutations in either/both proteins that will yield tighter binding. Of course, that could be very time consuming. Sarah Yohannan On 4/19/06, Wim Burmeister <[EMAIL PROTECTED]> wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Jerry McCully wrote: > > *** For details on how to be removed from this list visit the *** > > *** CCP4 home page http://www.ccp4.ac.uk *** > > > > > > Dear ccp4bb experts: > > > > > > I'm crystallizing a dynamic protein-protein complex. The two > > components bind to each other > > > > with a micro-molar affinity(This affinity was shown by Biacore3000 > > analysis, but I can not purify the complex by gel-filtration). > > > > > > > > > > I can get their crystals, respectively. But so far I have not > > > > got any crystals of the complex even if I tried several molar ratios > > between them. > > > > > > > > When I used the complex sample for crystallization, the smaller > > component was easily grown from the conditions containing PEG3350, and > > the other from MPD. > > > > > > > > Does anyone have the experience to crystallize this kind of > > low-affinity protein complexes? > > > > > > > > Any suggestion will be highly appreciated. > > > > > > > > Jerry McCully > > > > _________________________________________________________________ > > Is your PC infected? Get a FREE online computer virus scan from > > McAfee(r) Security. > > http://clinic.mcafee.com/clinic/ibuy/campaign.asp?cid=3963 > > > > > Dear Jerry > > usually the high protein concentrations encountered under > crystallization conditions will always drive the system to complex > formation (even for instable complexes with high dissociation > constants). Obviously, as well as some proteins do not crystallize some > complexes won't do so either. It should be sufficient to screen a high > number of crystallization conditions, usually the conditions of complex > crystallisation will be very different from the conditions of > crystallization of the individual compounds. I would be a bit worried > about the Biacore result, if it is a low micromolar dissociation > constant, I would expect that it should be possible to observe the > complex by gel filtration. It would be a good idea to try another method > for complex characterization (EM, microcalorimetry etc.) > > Yours > > Wim Burmeister > > -- > ******************************************************************************* > Wim Burmeister > Professeur, Membre de l'Institut Universitaire de France > Laboratoire de Virologie Moleculaire et Structurale FRE 2854 CNRS-UJF > c/o EMBL, 6 rue Jules Horowitz > B.P. 181, F-38042 Grenoble Cedex 9 FRANCE > E-mail: [EMAIL PROTECTED] > Tel: +33 (0) 476 20 72 82 Fax: +33 (0) 476 20 71 99 > http://www2.ujf-grenoble.fr/pharmacie/laboratoires/gdrviro > ******************************************************************************* > > >
