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Protein Crystallography Course
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Initial screening may be done by hanging drop vapour diffusion (protocol).
Commercially Available Screens
Hampton Research
Hampton Research make and distribute products for protein crystallization. Their kits have become the most common starting points for crystallizing previously uncrystallized protein.
Douglas Instruments have released a table for sorting the order of the Hampton conditions by type of precipitant.
For recording the data from the Hampton screens you can use Marko Hyvonen's tables.
They are based on the sparse matrix screening protocol.
- Hampton Research Crystal Screen
- Based on the method published by Jancarik and Kim
(Sparse matrix sampling: a screening method for crystallization of proteins. Jancarik, J. and Kim, S-H.J. Appl. Cryst. 24:409-411, 1991)
- 10ml each of 50 conditions
- In a survey by Hampton, conditions 25 and 27 were found to have not produced any crystals, so forget these two if you want to truncate the screen to 48 conditions to fit in two 24 well plates.
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Hampton Research Crystal Screen Lite
- Similar to Crystal Screen I but with the precipitant concentration halved.
- Gives lighter precipitate than the original crystal screen
- Hampton Research Crystal Screen Cryo
- Similar to Crystal Screen I but with the appropriate glycerol concentration for cryoprotection already added.
- The precipitant concentration is generally lower than Crystal Screen I, but not as low as Crystal Screen Lite
- Hampton Research Crystal Screen 2
- Based on the method published by Cudney et al.
(Screening and optimization strategies for macromolecular crystal growth. Cudney, B., Patel, S., Weisgraber, K., & Newhouse,Y, Acta Cryst D50:414-423, 1994)
- 10ml each of 48 conditions
- extension to Crystal Screen I
Hampton also make Grid Screens for popular precipitants.
Molecular Dimensions
Molecular Dimensions make the same kits, but the conditions are ordered by pH.
- Molecular Dimensions Structure Screen 1
- 10ml each of 50 conditions
- Molecular Dimensions Structure Screen 2
- 10ml each of 50 conditions i.e. two bonus conditions
- 49. 15% w/v Polyvinylpyrolidone
-
50. 2M Urea
- You can also order individual conditions eg. 500ml of condition 42.
3D Structure Screen
The 3D Structure Screen is a sparse matrix screen which givess 24 "nucleation" conditons and 24 "backed-off" conditions. In this kit the crystal growth trial is performed as a two step method. Firstly conditions are set up for nucleation and then the drops on their coverslips are transferred to wells with a lower concentration of precipitant for crystal growth. Saradakis & Chayen (Protein Science (2000), 9:755-757)
Clear Strategy Screens I & II
Clear Strategy Screens allow you to use the information already collected about the structure and activity of your protein to control folding homogeneity. The conditions are formulated at 90% of their final volumes allowing the starting pH to be changed depending upon your prior knowledge of the protein’s properties (e.g. isoelectric point, solubility/stability, previous experience with related proteins). The formulation principles also enable easy interpretation of results and simple planning of further experiments. Developed by Dr A M Brzozowski and J Walton at the University of York.
- Full control of screen solution pH.
- Cryoprotection of crystals.
- Rational planning of further experiments.
- Potential anomalous scattering centres.
Emerald Biostructures
Emerald Biostructures produce four kits for protein crystallization. They are sparse matrix screens like the Hampton Screens but have different conditions. Developed by Steve L. Sarfaty and Wim. G. J. Hol.
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Emerald Biostructures Wizard 1 Screen
- 10ml each 48 conditions
- final solution pHs between 4.2 and 10.5.
- 16 different precipitants
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Emerald Biostructures Wizard
2 Screen
- 10ml each 48 conditions
- companion to Wizard I
- same range of pH and precipitants
- Emerald Biostructures Cryo 1 Screen
- 10ml each 48 conditions
- All conditions will flash-freeze to a clear amorphous glass
- final solution pHs between 4.2 and 10.5.
- 13 different precipitants with 11 cryo=agents
- Emerald Biostructures Cryo 2 Screen
- 10ml each 48 conditions
- companion to Cryo I
- same range of pH and precipitants
Jena BioScience
Jena BioScience
produce 10 screening kits covering 240 conditions. Their compositions result from data mining of several thousands of
crystallized proteins. JBScreen represents the statistically most successful buffers that
yielded protein crystals suitable for X-ray diffraction.
(References: Jancarik & Kim (1991) J. Appl. Cryst. 24:409,
Gilliland et al. (1994) Acta Cryst. D50:408, &
Cudney et al. (1994) Acta Cryst. D50:414 )
- Each kit contains 24 buffer solutions, delivered in 0.7 ml sterile aliquots, with 10 units per kit.
- Screens ordered by type and concentration of the precipitant.
- JBScreen 1 (PEG 400 to 3000 based, including PEG MME)
- JBScreen 2 (PEG 4000 based)
- JBScreen 3 (PEG 4000 plus isopropanol, salt, glycerol &c based)
- JBScreen 4 (PEG 6000 to 8000 based)
- JBScreen 5 (PEG 8000 to 20000 based)
- JBScreen 6 (Ammonium Sulphate based)
- JBScreen 7 (MPD based)
- JBScreen 8 (MPD/Alcohol based)
- JBScreen 9 (Alcohol/Salt based)
- JBScreen 10 (Salt based)
- JBScreen Mixed kit contains one single shot of all the 240 buffers.
Other Screens
Other screens have been published but are not commercially available
- The Imperial College Screen
uses fewer pH values and more precipitants than the Hampton Grid Screen.
- The Palmerston North Screen uses PEG 6000 and PEG-mme 5000
at pH's ranging from 4.9 to 9.1.
- Michael Berry's thesis contains conditions for "A cheap, fast, and effective initial screen" as well as tables of effective precipitant salts, proven combinations of precipitants, and additives for perturbation screening
The Footprint screen includes the protein concentration as a parameter in the screen and maps out phase space.
The Reverse Screen
can be used when something is known about the crystallization conditions for the class to which the protein belongs. You make a guess of what are likely crystallization conditions rather than screening from scratch.
Reverse screening. E. A. Stura, A. C. Satterthwait, J. C. Calvo, D. C. Kaslow and I. A. Wilson. Acta Cryst. (1994). D50, 448-455
DIY Screens
- Online
- Crystool
is an online implementation of Brent Segelke's Crystool program, which generates a random screen.
- SAmBa
is a java applet that generates experimental protocols using orthogonal arrays of strength 2.
SAmBA: An interactive software for optimizing the design of biological macromolecules crystallization experiments
Audic S, Lopez F, Claverie JM, Poirot O, Abergel C, Proteins-Structure Function and Genetics 29: (2) 252-257 OCT 1997
- Downloadable software
- INFAC generates
incomplete factorial screens.
- GOSSET
generates minimum variance screens.
- XAct 1.0 for Windows 95/98. XAct is a stand-alone application for Microsoft Windows that can be used to construct, maintain, and record the results of many crystallisation experiments. Free to academic users.
- XtalGrow Software only for Intel x86 platform running Windows 95 or NT4
- Drop calculates the equilibration rate of a hanging drop experiment for NaCl and Ammonium sulphate.
Diller, D.J. and Hol, W.G.J. (1999) "An accurate numberical model for
calculating the equilibration rate of a hanging drop experiment". Acta Cryst.
D55, p. 656-663.
- Commercially available
Desalting
If your protein comes out of solution at low salt concentrations (i.e. needs a small amount of salt to stay in solution), desalting may be an alternative to salting-out.
Dialysing your protein against water is a good starting point.
Crystallizability is inversely proportional
to biological interest
Murphy's crystallization law
© 1999-2005 Airlie J McCoy,
University of Cambridge. All rights reserved.
Last updated:
7 June, 2005