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:[email protected]] On Behalf Of Damon
Colbert
Sent: Wednesday, February 04, 2009 8:08 PM
To: [email protected]
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: [email protected]
