1. © Johan Zeelen M.P.I. of Biophysics The crystallization of (membrane) proteins, using a flexible Sparse Matrix Screen. Soluble proteins and membrane proteins Screening protocol for 3 D crystallization - flexible sparse matrix - adjusted screen - optimisation Membrane proteins and detergent Excluding regions where nucleation is not evident

2. © Johan Zeelen M.P.I. of Biophysics Soluble proteins and membrane proteins - Soluble proteins Protein - Membrane proteins Protein Detergent (Lipid)

3. © Johan Zeelen M.P.I. of Biophysics Soluble proteins and membrane proteins The purity of the starting material is a crucial factor. - Purity of the protein: SDS gel and TLC - Sample heterogeneity: Mass spectrometry Dynamic light scattering - Solubility 5-10 mg/ml - Stability Storage conditions SDS - page- Thin-layer chromatography -> Proteolysis and post translational modifications -> Oligomerisation, aggregation

4. © Johan Zeelen M.P.I. of Biophysics Screening protocol for 3 D crystallization 12345678910 A B C D F G H I J 3.5Å 1.5Å. 8Å E Triosephosphate isomerase: P2 1 2 1 2 1 1.83Å C2 (big)2.1 Å C2 (small)2.1 Å P11.5 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

5. © Johan Zeelen M.P.I. of Biophysics Initial screen The systematic search of the multidimensional space requires large amount of protein and time. 12345678910 A B C D E F G H I J XXXX XXXX XXXX To reduce the number of crystallization trails, the incomplete factorial is a powerful tool to identify the influence of different variables. 12345678910 A B C D E F G H I J 123456789 A B C D E F G H I J X X X X 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. 12345678910 A B C D E F G H I J X XX X 123456789 A B C D E F G H I J 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. X XX

6. © Johan Zeelen M.P.I. of Biophysics Interpretation of the initial screen 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 precipitated Precipitated no birefringence no edges Precipitated with birefringence and edges

7. © Johan Zeelen M.P.I. of Biophysics No nucleation with the initial screen Try a different screen - random sampling - based on published conditions 0 1 2 3 4 5 34567891011 11 Commercially Available Screens Ammonium Sulfate pH 0 1 2 3 4 5 34567891011 Conditions from BMCD Ammonium Sulfate pH 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.

8. © Johan Zeelen M.P.I. of Biophysics No nucleation with the initial screen Adjust the initial screen Precipitant soluble supersaturation slow precipitation / nucleation Fast precipitation [Protein] No precipitate-> increase/double precipitant concentration Fast precipitation-> decrease/halve precipitant concentration 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.

9. © Johan Zeelen M.P.I. of Biophysics Optimisation screen 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 LiSO 4 at pH=6.5, 5 mg/ml protein -> small crystals after 3 days A) 12.5% - 25 % PEG 6000, 200 mM LiSO 4 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 LiSO 4 D) Protein 1 - 6 mg/ml, 25 % PEG 6000, 200 mM LiSO 4 at pH=6.5 A - B-> is salt needed A or B-> precipitant concentration C-> pH dependence D-> protein concentration Identical conditions -> reproducibility

10. © Johan Zeelen M.P.I. of Biophysics Detergent The detergent plays a crucial role in the crystallisation of membrane proteins. Neurospora crassa plasma membrane H + -ATPase precipitant detergent phase separation CMC 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.

11. © Johan Zeelen M.P.I. of Biophysics 2D and 3D crystallisation of LHC II 2D EM 3D X-ray

12. © Johan Zeelen M.P.I. of Biophysics Flexible sparse matrix screen Initial screen Adjusted screen Detergent/biochemistry OptimizationCrystals 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.

13. © Johan Zeelen M.P.I. of Biophysics Acknowledgements E.M.B.L Heidelberg Group R.K. Wierenga M.P.I. of Biophysics Department of Structural Biology W. Kühlbrandt H + -ATPase: M. Auer and G.A. Scarborough LHC II : M. Lamborghini http://www.mpibp-frankfurt.mpg.de/~johan.zeelen/xtal.html Garavito, R.M and Picot, D (1990) The Art of Crystallizing Membrane Proteins. Methods: A Companion to Methods in Enzymology Vol. 1 No. 1 pp. 57-69 Hjelmeland, L.M. and Chrambach, A. Solubilization of Functional Membrane Proteins. Methods in enzymology Vol 104 pp. 305-318 Zeelen, J.P. (1999) Strategy 1: A Flexible Sparse Matrix Screen. In Protein Crystallization, Techniques, Strategies, and Tips, chapter 9 (ed. T.M. Bergfors) IUL Biotechnology Series.