Section 24.4.6. Crystallization screens

With the introduction of the fast screen by Jancarik & Kim (1991), almost all attempts to crystallize a protein begin with experiments based on a screen of one form or another. The first screen was based on the ideas put forth by Carter & Carter (1979) in their discussion of the use of incomplete factorial experiments to limit the search, the experience of the investigators themselves, and the experience of others. A number of screens have been developed and even commercialized (e.g. Cudney et al., 1994). The first screens were quite general and applicable to a wide range of biological macromolecules, but fast screens based on specific classes of molecules such as RNA soon developed (Scott et al., 1995).

The BMCD is an ideal tool for facilitating the development of screens for general or specific classes of macromolecules. For example, if it were desired to produce a screen for endonucleases, a quick search of the PDB would provide the information in Table 24.4.6.1. An examination of the crystallization conditions of the endonucleases reveals that crystals are grown using a protein concentration ranging from 2.5 to 12.9 mg ml⁻¹, ammonium sulfate, sodium phosphate, or polyethylene glycol 400 to 8000 as precipitants at 4 to 20 °C between pH 4.5 and 8.3. A variety of buffers and standard biochemical additives are also used. From an examination of these parameters, a small subset of the crystallization experiments comprising an endonuclease screen could be developed.

Table 24.4.6.1| top | pdf | Crystallization conditions for endonucleases Endonuclease Crystal data (space group, unit cell) Crystallization method Protein concentration (mg ml−1) Chemical additives to reservoir pH T (°C) Reference(s) BamHI C2 76.4, 46.0, 69.4 Å, 110.5° Vapour diffusion with microseeding 12.5 10% Glycerol, 0.02 M potassium phosphate, 12% polyethylene glycol 8K 6.9 20 Newman et al. (1994) BamHI:12bp DNA 108.8, 81.9, 68.8 Å Vapour diffusion 10.8 5% Glycerol, 12% polyethylene glycol 8K, 0.15 M potassium chloride 6.9–7.6 22–24 Strzelecka et al. (1994) Crf10I, type II restriction I222 64.5, 81.3, 119.7 Å Vapour diffusion 6.0 1.0 M Ammonium acetate, 0.075 M MES 6.5–7.5 20 Bozic et al. (1996) EcoRV, type II restriction 58.2, 71.7, 136.0 Å Vapour diffusion 3.3–5.3 0.18 M Sodium chloride, 10% polyethylene glycol 4K 7.0–7.8 20–24 D'Arcy et al. (1985); Winkler et al. (1993) EcoRV, type II restriction:11-mer DNA P1 49.4, 50.2, 64.0 Å, 96.5, 109.1, 108.1° Batch 6.4–12.9 0.0043 M Phosphate buffer, 0.1073 M sodium chloride, 0.00040 M EDTA, 0.00040 M dithiothreitol, 0.0043 M cacodylate, 0.8–1.4% polyethylene glycol 4K 6.0–7.5 22–24 Kostrewa & Winkler (1995) EcoRV, type II restriction:cognate DNA 60.2, 78.4, 371.3 Å Vapour diffusion n/a 0.1 M Sodium phosphate, 0.08 M sodium chloride 6.4–6.9 19–22 Winkler et al. (1991, 1993) EcoRV, type II restriction:non-cognate DNA 68.4, 79.6, 6.4 Å, 104.6° Vapour diffusion 7.0–10.0 0.1 M Sodium chloride, 0.02 M MES 6.4–6.9 19–22 Winkler et al. (1991, 1993) EcoRV, type II restriction:product DNA P1 49.3, 50.3, 63.9 Å, 96.7, 108.8, 108.4° Microbatch 6.4 0.0043 M Phosphate buffer, 0.1073 M sodium chloride, 0.0004 M EDTA, 0.0004 M dithiothreitol, 0.0043 M cacodylate, 1–2% polyethylene glycol 4K, 0.0043 M magnesium chloride 6.0–7.5 4–22 Kostrewa & Winkler (1995) II, DNA repair [4Fe–4S] 48.5, 65.8, 86.8 Å Dialysis and macroseeding n/a 5.0% Glycerol, 0.0003 M sodium azide, 0.1 M sodium chloride, 0.005 M HEPES 7.0 15 Kuo, McRee, Cunningham & Tainer (1992); Kuo, McRee, Fisher et al. (1992) PvuII 84.2, 106.2, 46.9 Å Vapour diffusion 2.5 20–50% Saturated ammonium sulfate 5.0 18 Athanasiadis & Kokkinidis (1991) PuvII:cognate DNA 95.8, 86.3, 48.5 Å Vapour diffusion 9.6 0.0001 M EDTA, 2.5–3.6% polyethylene glycol 4K, 0.0155–0.0225 M sodium acetate 4.5 16 Balendiran et al. (1994) RuvC specific for Holliday junctions 72.8, 139.6, 32.4 Å, 93.0° Microdialysis 8.0 0.05 M TrisHCl, 7.5% glycerol, 0.001 M EDTA, 0.001 M dithiothreitol, 0.3–0.4 M sodium cloride 8.0 22–24 Ariyoshi et al. (1994) V, mutant E23Q 41.4, 40.1, 37.4 Å, 90.4° Vapour diffusion 10.0 0.05 M Potassium chloride, 0.008 M sodium cacodylate, 15% polyethylene glycol 400 4.5–8.0 4 Morikawa et al. (1995) V, mutant R3Q 41.4, 40.7, 37.4 Å, 90.1° Vapour diffusion 10.0 0.05 M Potassium chloride, 0.008 M sodium cacodylate, 15% polyethylene glycol 400 4.5–8.0 4 Morikawa et al. (1995) V 41.4, 40.1, 37.6 Å, 90.01° Vapour diffusion 10.0 0.05 M Potassium chloride, 0.008 M sodium cacodylate, 15% polyethylene glycol 400 4.5 4 Morikawa et al. (1988, 1992, 1995) V, mutant E23D 41.7, 40.2, 37.1 Å, 92° Vapour diffusion 10.0 0.05 M Potassium chloride, 0.008 M sodium cacodylate, 15% polyethylene glycol 400 4.5–8.0 4 Morikawa et al. (1995) Sm1 69.0, 106.7, 74.8 Å Vapour diffusion 10.0 0.01 M TrisHCl, 1.2–1.6 M ammonium sulfate 8.3 4 Bannikova et al. (1991) Extracellular 106.7, 74.5, 68.9 Å Dialysis 8.0 1.0–1.7 M Ammonium sulfate, 0.05 M sodium phosphate 6.0 4 Miller et al. (1991) Restriction, FokI:20pb DNA 65.6, 119.3, 71.5 Å, 101.4° Vapour diffusion with macroseeding 10.0 1.1 M Ammonium sulfate, 0.5 M MES, 0.2 M potassium chloride, 0.0005 M dithiothreitol, 0.0005 M EDTA, 5% glycerol 6.0 20 Hirsch et al. (1997); Wah et al. (1997)