Again you should read the spectacular paper by Obmolova and co. where they solved the structures of three Fab-antigen complexes using "MMS" microseeding (seeding into random screens), starting with one hit containing clusters of needles - which could not themselves be optimized
The paper is available on open access (free) http://journals.iucr.org/d/issues/2010/08/00/issconts.html http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf Acta Cryst. (2010). D66, 927-933 [ doi:10.1107/S0907444910026041 <http://dx.doi.org/10.1107/S0907444910026041> ] Promoting crystallization of antibody-antigen complexes via microseed matrix screening G. Obmolova <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Obmo lova%2C%20G%2E> , T. J. Malia <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Mali a%2C%20T%2EJ%2E> , A. Teplyakov <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Tepl yakov%2C%20A%2E> , R. Sweet <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Swee t%2C%20R%2E> and G. L. Gilliland <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Gill iland%2C%20G%2EL%2E> Synopsis: The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. Online 14 July 2010 The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: 10 September 2010 04:00 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
