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Re: AW: [ccp4bb]: membrane prot x-tal problem

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Speaking of cubic lipid phase crystallization, what detergent are you
using?  Do you know if your protein tends to oligomerize for function?
This might be important in CLP crystallization being that there is a
surface tension issue during incorporation of the protein into the CLP,
the smaller it is, the easier we believe it to start forming 2D crystals,
and eventually 3D.
 Bacteriorhodopsin is 26 Kda, and it forms trimers in CLP (as it does in
its native purple membrane).  These trimers are nicely separated and are
packed into one another via lipids.  As it turns out, these lipids are
those that were (surprisingly) carried over from expression in the native
bug, not the monolein used in the CLP setups.  We had success using a
variety of detergents for crystallizing BR in the CLP but not as much
success for larger proteins.  
 I also suggest you perform mass spec on the sample you're using, to make
sure you know what you have besides protein, ie. lipids if any.  Are you
expressing this in the native bug, purifying from tissue, or is it
recombinant from a host system?
 In your case of detergent crystallization, be wary of CMC's.

good luck to you


On Wed, 14 Jun 2000 Alexander.Pautsch@bc.boehringer-ingelheim.com wrote:
> *	how about other techniques as lipidic cubic phases (used for
> bacteriorhodopsin, Landau et al. in PNAS)
> > 
> > ***  For details on how to be removed from this list visit the  ***
> > ***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***
> > 
> > dear users,
> > 
> > i have recently beein trying to xtalize a 50kda membrane protein
> > and have had limited success. using peg 4000, and ammonium sulfate
> > i can get a few nice crystals. adding 1,6-hexanediol and some
> > alcohol improves this. however, the crystals dissolve almost as 
> > quickly as they appear, after one or two days. i've tried detergent
> > screens and additive screens but without success. does anyone know
> > how i might stabilize these xtals? any advise on membrane proteins
> > specifically would be a great help. 
> > 
> > stephen kelly
> > dept of microbiology
> > univeristy of alabama at birmingham
> > 
> > 
> > ***********************************
> > Songlin Li, Ph.D.
> > Dept. Microbiology
> > University of Alabama at Birmingham
> > AL35294, USA
> > Email: songlin@credit.cmc.usb.edu
> > ***********************************

Jean-Philippe Cartailler
Dept. of Molecular Biology and Biochemistry
University of California, Irvine
Hudel Lab, http://anx12.bio.uci.edu/~hudel/

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