On request, this summary (slightly amended) has been posted to the CPP4 wiki Crystal Growth page.
You can find it here: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality Regards, Damon. Damon Colbert schrieb: > > Thanks to everyone who responded with most helpful advice and > suggestions. I have provided a summary of the suggestions (and > clarifications to questions asked of me in return). > > Perma-Link to original question: > _https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=CCP4BB;AJMLIg;20090205 > 170801%2B1300_ > __________________________________________________________________ > > > * Concentrate protein with a higher molecular weight cutoff (e.g. > 50-100 kDa). > > * Protein is known to form a tetramer, and by approximation from gel > filtration elution, exists as a 126 kDa species (~114 kDa tetramer > and ~22 kDa OG micelle). It usually elutes as a single, > well-resolved peak (unless, for example, I am using it to exchange > detergent). DLS has shown monodispersity in samples, but I don't > use it routinely. > > > > * Dialyse protein overnight (routinely or after centrifugal > concentration) to reduce and define the detergent concentration. > > * This can get expensive, using relatively large volumes detergent > to make the dialysis buffer. Nonetheless the most recent crystals > were obtained from dialysed protein. > > > > * Trial extraction, purification, and crystallisation with different > detergents (using desalting or Q-sepharose columns). Poor > diffraction could be indicative of detergent-mediated crystal > contacts (rather than protein-protein). > > * Use of shorter detergents (e.g. Cymal-3 to -6) or mixed detergent > micelles > * Reconstruct sparse matrix screens with each different detergent > * See Lemieux /et al/. (2003), Protein Science. > > > > * Identify membrane lipids associated with protein (in-house by TLC > or otherwise). Retaining some native lipid or adding it back in > at crystallization may improve crystal quality. Conversely total > delipidation may also help. > > * Need to correlate successful crystallisation with presence/absence > of lipid > * Could try using lipid-like detergents (FC or DHPC) > > > > * Deglycosylation is checked on SDS-PAGE, and confirmed by the loss > of higher molecular weight smears (which are present before > deglycosylation reaction). > > * Alternatively protein could be digested with Endolgycosidase H, > which leaves one GlcNac residue on each glycosylation site. This > could improve crystal contacts. See Chang, V.T. /et al/. (2007) > "Glycoprotein structural genomics: solving the glycosylation > problem." Structure 15(3):267-73 > > > > * Chemical modification of surface residues may improve crystal > contacts, for example lysine methylation. > > * See Walter /et al/. (2006) "Lysine methylation as a routine rescue > strategy for protein crystallization." Structure 14(11):1617-22 > * Mutagenesis is another alternative, but we have not yet been > successful producing a recombinant protein. > > > > * Adding salt (or PEG) to reservoir solution may promote crystal > growth in the aqueous phase, rather than the 'oil/gel' phase. > > * Conditions producing the crystals grown in this 'gel' had PEG 1K > or 2K as precipitant, and low [NaCl] present. (Is the suggestion > 'to increase the concentration beyond that of the reservoir > solution?'). > > > > * Test crystallisation conditions at low temperature (e.g. 4°C) > > > > * Test oils (paraffin or paraton-N) as cryoprotectants. > Alternatively maintain detergent concentration in cryoprotectant. > > * Paratone oil (softened with some mineral oil) was used with poorly > diffracting native crystals, and showed no improvement in > diffraction. It has not been attempted with more recent protein > crystals grown in presence of ligand. > > > > * Attempt to collect a 10Ang dataset and try MR with a close homolog. > > > > Many thanks. > > Regards, > Damon.
