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P212121 1.83ÅC2 (big) 2.1 ÅC2 (small) 2.1 ÅP1 1.5 Å
The screening for suitable crystallization conditions starts with the search in a multidimensional phase diagram for conditions that favor nucleation. Experiments with Triosephospate isomerase (soluble protein) and Light-harvesting complex II (membrane protein) indicate, that the proteins can crystallize in different space groups and resolution.
Two dimensional representation of the multi dimensional crystallization space
Initial screenThe systematic search of the multidimensional space requires large amount of protein and time.
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To reduce the number of crystallization trails, the incomplete factorial is a powerful tool to identify the influence of different variables.
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The conditions in the sparse matrix are not random but heavily biased towards published crystallization conditions. Most of the commercially available screens are sparse matrix screens.
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In the flexible sparse matrix also information from biochemistry (pH stability, salts) is used to exclude conditions where the protein denatures / is inactive.To simplify the interpretation of the results, the crystallization conditions are sorted by precipitant. The drop size is 1 µl protein and 1 µl well solution.
The crystallization experiments are examined with a stereo microscope: 1) immediately after setup, 2) each day for the first week, and 3) once a week for several weeks.
Not precipitatedPrecipitated no birefringence no edgesPrecipitated with birefringence and edges
As initial screen the sparse matrix is the most popular because it is commercially available. This does not mean that every protein will crystallize under these conditions. The strategy to try different sparse matrix screens will limit the search to the most common successful crystallization conditions.
Repeat until the precipitation point for all wells is determined.
A different and possibly faster strategy is to use the solubility information from the first screen to exclude areas of the phase diagram where no crystallization will occur. This knowledge is used for the design of a new adjusted screen. In the adjusted screen only the precipitant concentration is changed.
After identification of conditions that favour crystal growth a new set of conditions is set-up by adjusting the:
A) precipitant 1 concentration with salt
B) precipitant concentration without salt
C) buffer and pH (4.5-9.5)
D) protein concentration
Initial screen
Adjusted screen
Optimisation
25 % PEG 6000, 200 mM LiSO4 at pH=6.5, 5 mg/ml protein -> small crystals after 3 days
A) 12.5% - 25 % PEG 6000, 200 mM LiSO4 at pH=6.5 B) 12.5% - 25 % PEG 6000 at pH=6.5 C) pH = 4.5 - 9.5, 25 % PEG 6000, 200 mM LiSO4 D) Protein 1 - 6 mg/ml, 25 % PEG 6000, 200 mM LiSO4 at pH=6.5
A - B -> is salt neededA or B -> precipitant concentrationC -> pH dependenceD -> protein concentrationIdentical conditions -> reproducibility
Detergent phase diagram At high detergent and precipitant concentrations the micelles aggregate. The solution separates in a micelle rich and a micelle poor phase. Often 3D crystals are found, close to the condition where phase separation occurs.
The flexible sparse matrix is an efficient method for searching the multidimensional phase diagram for conditions that favor nucleation, by excluding regions where nucleation is not evident. Although the preparation of wells from stock solutions is more labor intensive than ready-made solutions, it has the advantage that precipitant concentration, pH, and additive can be manipulated independently.