Dear CCP4bb, I am attempting to crystallise a 25kDa membrane protein of eukaryotic origin. We have obtained crystals of the protein (with and without a potential ligand). However crystal quality is poor, as exposure at room temperature and cryo-protected conditions have given diffraction as far as 10-15Ang at best. After scouring the CCP4bb archives and local expertise, we have not yet had much success in improving crystal quality, and wished to probe the knowledge of the community for additional ideas. __________________________________________________________________________________________________________________________________________
The background; The protein is purified from its native source, solubilised with octyl-glucoside detergent, and treated with a recombinant PNGaseF to remove glycosylation. After an initial Q-sepharose and size exclusion chromatography, the protein is concentrated by elution from a small volume of Q-sepharose resin (centrifugal concentration is occasionally used, but introduces an unknown in the detergent concentration, the monomers of which move through the concentrator membrane, but not micelles). The protein has been reproducibly crystallised in glycine and bicine buffers, at low pH ( 9.0-10.0 ), low molarity salt, and 30-33% PEGs of various molecular weights (e.g. PEG 300, 1000, 2000). Native crystals had a size and morphology very similar to crystals of a close homologue, appearing sharp-edged and quite stable to careful manipulation with cryo-loops. Crystals obtained in the presence of the potential ligand had a different, less sharp morphology (more like thin plates). Notably the latter crystals seemed to form only in what appeared to be a phase separation, but on manipulation seemed more like a gel, perhaps protein precipitate. The gel made the crystals difficult to manipulate, and possibly resulted in mechanical damage. Of more concern I believe it may have been looped up with the crystal and prevented proper cryoprotectant penetration, although there were no ice rings to indicate so. Neither form diffracted beyond 10Ang, even in different cryoprotectants (higher PEG concentration, 25% glycerol, sucrose, and ethylene glycol). Furthermore automated annealing (for 1sec) did not improve diffraction. Seeding has been trialled for the native crystals (not yet for ligand-bound forms), but has not improved crystal growth. The purification detergent used is being reconsidered. We have previously attempted to crystallise the protein in nonyl-glucoside detergent, without success. Various additive detergents (below their CMC), alcohols and other amphilic additives have been screened, without success in crystallisation. We aim to swap the protein into different detergents (i.e. maltosides) and try for improved crystal quality under known conditions. We are also considering crystal dehydration, in an attempt to reduce solvent content. Additionally I have attempted reproducing conditions with 0.1% w/v agarose as an additive, aiming to promote growth of the latter crystal form without the difficult gel phase. Finally I have toyed a bit with cubic lipid phase crystallisation, without any success so far. Any advice on these specific approaches would be most appreciated. As you can see we have considered many methods. If there is something I have missed, or perhaps some common pitfall I have not anticipated, I would appreciate any advice you have to offer. I thank you for taking the time to read this mini-essay, and again for answering my off-topic request for advice. Regards, Damon. __________________________________________________ Damon Colbert School of Biological Sciences University of Auckland Email: [email protected]
