Re: [ccp4bb] SUMMARY: Poor diffraction of eukaryotic membrane protein crystals
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.
[ccp4bb] SUMMARY: Poor diffraction of eukaryotic membrane protein crystals
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;20090205170801%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 1 Reconstruct sparse matrix screens with each different detergent 2 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 1 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 1 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. __ Damon Colbert School of Biological Sciences University of Auckland Email: d.colb...@auckland.ac.nz
