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Subject:

Re: suggestion of crystallization optimization

From:

Janet Newman <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Mon, 4 Jun 2018 20:41:34 +0000

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Liuqing Chen,

Everything that has been said seems reasonable, but there are always infinite possibilities in crystallisation, so it is more a question of priorities. Do the easy (or quick) things first. If you have buckets of prepared protein then what you will try first might be different than if you have to go and make your protein from scratch each time you set up crystal trays.

1. If you have crystals from an additive screen or seeding - try putting them in the beam. If you have access to in-plate screening, you can test the crystals without disturbing them, which will give the best idea of their native diffraction. Perhaps one of the ugly crystals diffracts well enough?

2. Try cross seeding - seed one or more initial screen(s) (rather than an optimisation).  Try initial screening with seeding at different temperatures. If you are currently using vapour diffusion, try microbatch. Or vice versa.

3. Try in-situ proteolysis. Add a very small amount of protease to your protein sample (chymotrypsin is traditional) - say 1:10,000 or 1:1000 compared to your protein concentration then set up that mixture in initial screens. 

4. Is there a ligand/inhibitor/other small molecule that binds? Add that to your protein before crystallisation. Maybe even try this first!

5. Lysine methylation/cysteine modification/other side chain modifications.

6. Try using DSF or some other technique to look at your protein's stability in the formulation it is in. Maybe you can make happier protein by changing the pH, buffer or salt.

7. If you want to be a little more rigorous, take your protein, and a number of different proteases, and do a time-course experiment with each protease (add 1:1000 protease to your protein, then take samples at timepoints - say 0.5 hours, 1 hour, 5 hours and overnight) then run out on a gel (or analyse by MS) and see if you come down to a stable fragment. If you do - then use that protease, and while you are waiting for the crystallisation trials to do their thing, find out what the end points of the proteolysis fragment are, and make that construct.

6. Try a different expression system (different tag, different position of the tag, cleave/don't cleave the tag). If the protein is produced in a eukaryotic system (and is glycosylated) try a different one to get different glycosylation pattern. Try kifunensine treated cells if you are in a mammalian expression system.

8. Try the same protein from other species

Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email [log in to unmask]

________________________________________
From: CCP4 bulletin board <[log in to unmask]> on behalf of Liuqing Chen <[log in to unmask]>
Sent: 04 June 2018 20:57
To: [log in to unmask]
Subject: [ccp4bb] suggestion of crystallization optimization

Hello everyone!

I get a crystal several months ago, but the crystals diffraction very low resolution (around 8A)  or no diffraction.
  My sample buffer is 20mm Hepes ph7.0,  50mm NaCl,   my protein pI is 5.
  the codition grow crytal  is : 30% PEG400, 100mm hepes 7.5,  200mm MgCl2.

I also tried  additive screen,  all the crystals appear the same apparence,    even i seeding optimization,  have no improve.
the  attach is  my crystals.

what should   i  do next?

thanks in advance.
sincerely
Liuqing chen

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