| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | Advert | | |
Re: [ccp4bb] Poor diffraction of eukaryotic membrane protein crystals |
||
- Protein crystallographyMain steps:- Protein purification- Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Poor diffraction of eukaryotic membrane protein crystals From: "Das, Debanu" debanu {- at -} SLAC {- dot -} STANFORD {- dot -} EDU Date: 2009-02-05 Hi Damon, In case your target oligomerizes to produce an assembly larger than 100 kDa, you may try to use the 100 kDa MWCO concentrator which will not only allow the detergent monomers to pass through, but also the micelles for most detergents used for membrane proteins. The 50 kDa one will also allow micelles of some detergents to pass through. In addition to trying different detergents as additives, you might consider doing a) extraction and purification/crystallization with numerous different detergents; b) detergent exchange at the last purification stage. i.e, the SEC. How pure is the target under current pufification protocols? You may need to purify more. Send your purified sample for lipid analysis, identify what kind of native lipids are present in membrane proteins in the system you are working with and try to supplement such lipids in the purification if you are missing any. Sometimes total delipidation may help (which can be stripped off during purifcation) and sometimes you may be better off with native lipids. Also look into some of the lipid-like detergents like FC and DHPC. You can also try TLS in-house to check for lipids in your sample. You can try to clone into appropriate vectors for expression in other hosts like E. coli or yeast which would allow you to easily try several different constructs which may improve resolution of the crystals. You may also try working with other close homologs. If there is a close homolog whose structure has been determined, you may try to collect a full data set at 10A and try molecular replacement. I have tested MR using PHASER on 120 kDa membrane protein to find the lowest resolution at which the solution could be found, and it was ~12A (this was done with a 3.0A complete data set but MR search cut back to different low res values). However, in this test case, the search model used was the structure of the same protein, so may be that's why it worked out. But it never hurts to try. Make sure you are trying out as much of the membrane protein crystallization screens that are available if you have access to a crystallization robot. Have you checked that the target was properly deglycosylated? Enzyme efficiences vary based on detergent type and concentration. I suppose you have checked for this already. If your crystals are large enough and you have many of them that you can pick up and wash well, it would be best to try to get a mass spec on them or run them on a gel to verify. You may try the Rigaku FMS system if you have access to a synchrotron beamline with such a setup. Regards, Debanu. ___________________ Debanu Das, JCSG, SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA. ________________________________ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Damon Colbert Sent: Wednesday, February 04, 2009 8:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [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.colbert@auckland.ac.nz3 CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
|
| ProteinCrystallography.org: Copyright 2006-2010 by Quid United Ltd |