Hello again, I still think it depends on the protein complex, but I agree that the consensus/ hearsay/ anecdotal/ old crystallographer's tales revolve around complexes not working as well as single proteins for freezing. I'm not sure that has been shown to be the case, although again I would agree that it is hard to prove anything like this and one would just have to take a statistical approach and say for X% this has happened.
I guess my points are: 1) you have to freeze/thaw the right way, 2) an 'optimized' buffer system for the complex or even single proteins should be used, 3) if you are looking for a single crystal and that is all you need, then fresh preps are great. If you will need more than just one single crystal, reproducibility matters, and in the cases I've worked on, freezing is almost always more reproducible. If in the case posted, the freezing was done in an Eppi tube, by putting it in the freezer, with a non-optimal buffer and then just pulled out and put into an ice bucket- I would expect almost every protein/complex to fall out of solution. If instead the buffer had been optimized, the protein snap frozen and snap thawed in a thin walled tube, then maybe one needs to set up the protein in crystallization trials as soon as one has purified the complex and not freeze or wait around. Of course, the caveat is that one should never believe generalizations... Cheers, tom ________________________________ From: David Briggs [mailto:[email protected]] Sent: Thursday, 22 April 2010 6:07 PM To: Peat, Tom (CMHT, Parkville) Cc: [email protected] Subject: Re: RE: [ccp4bb] degradation of protein durring freez thaw Ouch! I completely agree re: single proteins, but I had always found/heard that protein-protein complexes tolerate freezing/thawing less-well that their individual components. Irrespective of generalisations, In the posted case, where apparently, before freeze complex is intact and concentrated, after freeze complex is less concentrated and degraded, surely the most straight-forward fix is to not freeze it? Dave -- Delivered via an Android. On Apr 22, 2010 8:57 AM, <[email protected]> wrote: Hello- Sorry to be disagreeable, but I think it depends on the protein. We've had much better success in getting reproducible crystals when we've snap frozen in liquid nitrogen in small aliquots (thin walled PCR tubes work best). We can then screen and optimize using the same protein prep- and again, it is much more reproducible than doing a fresh prep each time. Of course this is only for the few hundred proteins I've worked on and it has only been for 98% of the cases examined. Cheers, tom ________________________________ From: CCP4 bulletin board [mailto:[email protected]<mailto:[email protected]>] On Behalf Of David Briggs Sent: Thursday, 22 April 2010 5:27 PM To: [email protected]<mailto:[email protected]> Subject: Re: [ccp4bb] degradation of protein durring freez thaw Hi, Obvious answer - don't freeze it. If you cannot set your crystallisation screens up straig...
