Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
I have had a lot of problems of my own with poorly diffracting (or not at all) membrane protein crystals. After a previous discussion here, I summarised the suggestions I got in this ccp4 user wiki; http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality There are a few ideas there that have already been put forward. My personal opinion is that (assuming your crystals are protein) detergent is the deciding factor. I second the suggestion for shorter chain detergents, but also suggest that you carefully consider your detergent concentration. While you want it to be above cmc, I have found excessive concentration to be bad for crystallisation. If you are going back to the drawing board, I can highly recommend the MemGold and MemStart/Sys screens developed by the lab of So Iwata at the Imperial College, and sold by Molecular Dimensions (in the UK). It has given me a lot of success in getting initial hits for various membrane proteins. MemGold has been designed to specifically target alpha-helical membrane proteins, as described in this paper; http://onlinelibrary.wiley.com/doi/10.1110/ps.073263108/full Hope you find something useful here. Good luck. Damon.
Re: [ccp4bb] linux question
I have a persistent Kubuntu 8.10 on a 16Gb flash drive, on which I have been running the likes of Coot, iMosflm and CCP4. I have used my setup to process two datasets via MolRep. There are plenty of resources on the internet telling you how to set it up. e.g http://www.pendrivelinux.com/ http://www.linux-usb.org/ https://wiki.kubuntu.org/LiveUsbPendrivePersistent It was relatively quick to set up, but it took a week to work out some problems. The main one being that with limited space on the thumb drive, one cannot do a complete update of Kubuntu. Therefore many libraries are missing and dependancy errors crop up during crystallography software installation. It's not too difficult a problem to deal with if you are familiar with installing apps on linux. And by partitioning your drive with FAT32, and two ext3 partitions (one for the root, and one labelled casper-rw) before creating the linux boot-stick, you can have a bootable linux usb stick with the ability to share files with a windows machine (fat32 partition) and enough space to install those behemoth crystallography apps. And I managed to do all this with minimal linux experience. (The only issue I haven't yet resolved is with graphics drivers. Its a trivial task to install the appropriate drivers, but it seems every time I boot up Kubuntu, I have to run 'x-config' and restart the x-server to get the Nvidia drivers to work, which is necessary for Coot, at least. An inconvenience every time I start linux, but not impossible). Im not familiar with setting up or running virtual machines / emulators, but they might be a simpler, more versatile option? But at least you can run graphical applications with ease (pymol and coot run well once the nvidia drivers are working).
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
[ccp4bb] Poor diffraction of eukaryotic membrane protein crystals
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: d.colb...@auckland.ac.nz3
