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Kate Augustyn 04-03-2009 11:55 PM

Protein crashes out in crystallography solution
I am trying to crystallize a protein inhibitor complex and am adding 1:1 protein (5 mg/ml, 2.5 mg/ml, 1.25 mg/ml) with 5X inhibitor to a crystallization solution containing various combinations of LiSO4, NaOAc, NaCl or Hepes, LiSO4, conditions which I found produced crystals from an initial screen. However, when I try and repeat these conditions on a larger scale it appears the protein crashes out as soon as it comes in contact with the crystallization solution. Does anyone know if it is possible to have the protein go back into solution and then crystallize or is it a lost cause?

ted9999 04-15-2009 04:08 AM

Re: Protein crashes out in crystallography solution
Yes it is possible, check out the following paper:

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ConsultEnzy 07-19-2009 11:47 AM

Re: Protein crashes out in crystallography solution
As the previous user said, yes it is very possible. It could even be your initial crystals grew back from such precipitates?

Anyway, by now you should have resolved the problem one way or another, but if not, have you ever tried macro- or micro-seeding?

I say this as it could be seeding into your precipitates may lead to rapid crystal growth, whereas just waiting around may not lead to any nucleation events.

The reason your drops are behaving differently to your initial screen could be numerous, such as:

1. Slightly different temperature
2. You took longer to set them up in different humidity
3. You made up the crystallisation solutions instead of using them from the commercial screen, and things ended up slightly different
4. etc etc

However, the major difference is you added your inhibitor! This potentially changes things so much one should expect to go back to square one and screen from the beginning....

However, after saying that I would say you stand a good chance of seeding into your identified lead conditions (that contain your inhibitor) and getting crystals.

Don't spend ages trying to do perfect micro-seeding initially.....just add big messy chunks of freshly smashed up crystal into the precipitated protein/inhibitor mix (one quick streak through using a loop) and get the slips turned back over. I would say you may see growth the next day for one of your conditions - fingers crossed - good-luck!

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