The BCS Screen
PEG smears for broader chemical space coverage
The BCS (Basic Chemical Space) screen developed at the Structural Genomic Consortium (SGC), Oxford has a novel approach to screening. Based on idea from Newman et al, 2006, PEGS are grouped by molecular weight and mixed to create four PEG smears (available to purchase separately). The smears cover a broader range of chemical space while reducing the number of PEG variables.
PEG is an effective crystallisation agent due to its protein-protein interaction promoting properties. The Biological Macromolecular Crystallization database records at least 20 different PEG compounds in frequent use as macromolecular precipitants. The BCS screen overcomes the impracticality of sampling all PEG varieties that has led to a few variants predominating in the most common screens (right).
The bottom line is that in tests at the SGC with 191 human proteins, the BCS screen had a 42% hit rate with only 96 conditions. Many of these proteins had failed to crystallize in other screens.
Some crystals grown in the BCS screen are shown on the left. Phosphoglycerate mutase 5 and ETV1/DNA complex ONLY crystallize in the BCS screen. Crystals of MMAA, CDKL5, RASSF3 grown in this screen diffract better than those grown from other screens. Novel crystal forms of UGDH, p38α/TAB1 complex, ERK2/VTX-11e inhibitor grew in the BCS screen.
Publications arising from crystals grown using this screen include: Froese, DS et al. J Biol Chem 285:38204 (2010); Chaikuad, A et al. PNAS 108: 21029 (2011); Egger, S et al. J Biol Chem 287:2119 (2011); Filappakopoulus, P et al. Cell 149: 214 (2012); Chaikuad, A et al. Nat Chem Biol 10: 853(2014); Kvackova, S et al. J Med Chem 58: 3393 (2015); Drouin, L et al. J Med Chem 58: 2553 (2015); Chaikuad, A et al. J Med Chem 59: 1648 (2016); Chen, P et al. J Med Chem 59: 1410(2016).
Single PEG or Smear?
PEG smears consist of a mixture of PEG compounds. The BCS screen utilises four different smears (see right) - low molecular weight, medium molecular weight, high molecular weight and broad range of molecular weights. In each case equal volumes of individual stock solutions at equal concentrations are mixed to produce the final solution.
PEG smears are designed primarily for efficient screening of chemical space. Received wisdom says that well-defined, single PEG species produce the best diffracting crystals. Thus, optimization might involve identifying the single PEG compound that produces the best crystal for structural studies.
HOWEVER, in some cases the use of a PEG smear from the BCS screen produced better quality data than using any single PEG species. On the left, you can clearly see that MMAA crystals grown with a LMW smear diffract better than those grown with a single PEG species.
The individual PEG smears available to purchase separately for further optimization.
How it works
The first 24 conditions in the BCS screen form a grid screen of the four PEG smears against pH (range 4.5-9.5). The remaining 72 conditions consist of a sparse matrix screen in which the precipitant is always one of the PEG smears but with a variety of additives and buffers. The additives include some of the more unusual crystal-promoting agents such as RbCl (also useful for phasing) and ammonium nitrate. More common additives including NaBr (useful for phasing) and glycerol are also used in the screen.
Chaikuad, A, Knapp, S and von Delft, K (2015). Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens. Acta Crystallographica F71: 1627-1639.
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