Difference between revisions of "Crystal growth: Tips and Tricks"

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== I tried Hampton Screen I & II with my protein and have no crystals.  What can I do? ==
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==Getting crystals==
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===I tried Hampton Screen I & II with my protein and have no crystals.  What can I do?===
  
1.  There are other screens.  While it is proably true that when proteins do crystalize, they in many cases produce hits from Hampton screens I or II, we are dealing here with exception to that rule.  Lists of screens are available from manufacturer websites:
+
# There are other screens.  While it is proably true that when proteins do crystalize, they in many cases produce hits from Hampton screens I or II, we are dealing here with exception to that rule.  Lists of screens are available from manufacturer websites:
 +
#* [http://www.hamptonresearch.com/ Hampton Research]
 +
#* [http://www1.qiagen.com/Products/Protein/Crystallization/ScreeningSuitesInPreFilledFormats/NeXtalTubes.aspx Qiagen (Nextal)]
 +
#* [http://www.emeraldbiosystems.com Emerald Biosystems]
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#* [http://www.jenabioscience.com/cms/en/1/browse/632_screens.html Jena Bioscience]
 +
#* [[Crystallization screens and methods | Other screens ]]
 +
# Vary protein concentration.  This is rather obvious suggestion when all your drops are clear or all have precipitate, in which case your overall concentration is too low (too high).  More elaborate approach is to modify concentrations based on the results of your first screen, i.e. double it for the conditions which produced clear drops and cut it in half for those which produced precipiate.  To avoid concentrating/diluting protein, you can simply try to mix your original protein stock with reservoir solutions in different proportions.  For instance, mixing 2ul of protein with 1ul of reservoir solution is (to some degree) equivalent of doubling protein concentration.
 +
# Set your screens at different temperature.  25 and 4 degrees are the most popular options.  If you completely lost hope for your original screens, just transfer them to the cold room.  Protein solubility will change and you may get crystals.
 +
# Add a purification step. Purity can be critical to protein crystallization and the purity  judged from SDS-PAGE or even gel filtration can be misleading.
 +
# [[Modifying_the_protein_to_crystallize_better | Modify your protein. ]]
  
- [http://www.hamptonresearch.com/ Hampton Research]
+
==Improving crystals==
 
+
===Reducing the mosaicity===
- [http://www1.qiagen.com/Products/Protein/Crystallization/ScreeningSuitesInPreFilledFormats/NeXtalTubes.aspx Qiagen (Nextal)]
+
* incubate your crystal longer in your cryo/stabilization solution. This helps sometimes to lower
 
+
mosaicity. Of course, you can also try co-crystallization with glycerol (2%, 5% or 10%).
- [http://www.emeraldbiosystems.com Emerald Biosystems]
+
* the mosaicity may be anisotropic, so try to orient the crystal in the loop such that you can shoot along a different axis
 
+
* look at Elspeth Garman's papers, and/or check her talks at RapiData workshops (anybody with a link??)
- [http://www.jenabioscience.com/cms/en/1/browse/632_screens.html Jena Bioscience]
+
* change precipitant, pH, temperature, and optimize cryo conditions
 
 
- [[Crystallization screens and methods | Other screens ]]
 
 
 
2.  Vary protein concentration.  This is rather obvious suggestion when all your drops are clear or all have precipitate, in which case your overall concentration is too low (too high). More elaborate approach is to modify concentrations based on the results of your first screen, i.e. double it for the conditions which produced clear drops and cut it in half for those which produced precipiate.  To avoid concentrating/diluting protein, you can simply try to mix your original protein stock with reservoir solutions in different proportions.  For instance, mixing 2ul of protein with 1ul of reservoir solution is (to some degree) equivalent of doubling protein concentration.
 
 
 
3.  Set your screens at different temperature.  25 and 4 degrees are the most popular options.  If you completely lost hope for your original screens, just transfer them to the cold room.  Protein solubility will change and you may get crystals.
 
 
 
4.  Add a purification step. Purity can be critical to protein crystallization and the purity  judged from SDS-PAGE or even gel filtration can be misleading.
 
 
 
5.  [[Modifying_the_protein_to_crystallize_better | Modify your protein. ]]
 

Revision as of 19:15, 3 June 2008

Getting crystals

I tried Hampton Screen I & II with my protein and have no crystals. What can I do?

  1. There are other screens. While it is proably true that when proteins do crystalize, they in many cases produce hits from Hampton screens I or II, we are dealing here with exception to that rule. Lists of screens are available from manufacturer websites:
  2. Vary protein concentration. This is rather obvious suggestion when all your drops are clear or all have precipitate, in which case your overall concentration is too low (too high). More elaborate approach is to modify concentrations based on the results of your first screen, i.e. double it for the conditions which produced clear drops and cut it in half for those which produced precipiate. To avoid concentrating/diluting protein, you can simply try to mix your original protein stock with reservoir solutions in different proportions. For instance, mixing 2ul of protein with 1ul of reservoir solution is (to some degree) equivalent of doubling protein concentration.
  3. Set your screens at different temperature. 25 and 4 degrees are the most popular options. If you completely lost hope for your original screens, just transfer them to the cold room. Protein solubility will change and you may get crystals.
  4. Add a purification step. Purity can be critical to protein crystallization and the purity judged from SDS-PAGE or even gel filtration can be misleading.
  5. Modify your protein.

Improving crystals

Reducing the mosaicity

  • incubate your crystal longer in your cryo/stabilization solution. This helps sometimes to lower

mosaicity. Of course, you can also try co-crystallization with glycerol (2%, 5% or 10%).

  • the mosaicity may be anisotropic, so try to orient the crystal in the loop such that you can shoot along a different axis
  • look at Elspeth Garman's papers, and/or check her talks at RapiData workshops (anybody with a link??)
  • change precipitant, pH, temperature, and optimize cryo conditions