A drug-discovery-oriented non-invasive protocol for protein crystal cryoprotection by dehydration, with application for crystallization screening

Abstract

In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitro-gen or propane. Protocols for protein crystal cryoprotection are based on either increasing the environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13 M potassium formate (KF13), directly to the reservoir. Several salt solutions were screened to identify KF13 as optimal. Cryoprotection using the KF13 protocol is non-invasive to the crystal, high throughput and easy to implement, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug-discovery projects which use very large compound or fragment libraries. An application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.

Keywords: cryoprotection; crystals; dehydration; high throughput; proteins.

Figure 1

Schematic drawing of the procedures involved in crystal drop dehydration using the KF13 protocol.

Figure 2

Dehydrating effects on crystallization drops from significantly different conditions after 24 h from adding KF13 to the reservoirs. Initial drop volume was 200 nl of reservoir plus 200 nl of a protein-less solution containing 500 mM NaCl. The conditions are from a crystallization plate from our facility and contain 2.5 M ammonium sulfate, 0.2 M NaCl and 0.1 M MES pH 5.6 (A10); 1.2 M ammonium sulfate and 0.1 M MES pH 5.9 (A11); 14% ethanol and 0.1 M ADA pH 6 (B8); 18% ethanol and 0.1 M bis-tris propane pH 7.1 (B9); 15% PEG 2K (w/v) and 0.1 M bis-tris propane pH 6.9 (C10); 8% PEG 20K, 8% PEG 2K (w/v), 0.25 M KBr and 0.1 M sodium acetate pH 4.5 (C11); 21% PEG 3350, 0.15 M NaCl and 0.1 M MES pH 6 (D6); 15% PEG 3350 and 0.1 M MES pH 6.2 (D7); 28% PEG 400, 0.2 M NaCl and 0.1 M MOPS pH 6.5 (E7); 25% PEG 400, 4.5% ethanol, 1.5 mM MgCl₂ and 0.07 M MES pH 6.6 (E8); 14% PEG 4K, 6% MPD and 0.1 M sodium potassium phosphate pH 6.2 (F4); 29% PEG 4K, 0.1 M ammonium sulfate, 0.1 M magnesium acetate and 0.1 M sodium citrate pH 6.5 (F5); 13% PEG 8K, 0.09 M ammonium sulfate and 0.05 sodium cacodylate pH 6.5 (G8); 20% PEG 8K, 0.2 M magnesium acetate and 0.1 M MES pH 6.5 (G9); 1 M potassium phosphate monobasic, 3% iso­propanol and 0.1 M sodium cacodylate pH 6.5 (H1); 1.4 M sodium acetate and 0.1 M sodium cacodylate pH 6.5 (H2).

Figure 3

Diffraction images from crystals of FtsA filaments dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown.

Figure 4

Diffraction images from crystals of GluLBD in complex with agonist dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown.

Figure 5

Diffraction images from crystals of the hetero-pentameric complex Cenp-OPQUR dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown.

Figure 6

Diffraction images from crystals of lysozyme dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown. The number after the sample name indicates the amount of precipitant that was used in crystallization (see Materials and methods and Table 1 ▸).

Figure 7

Diffraction images from crystals of concanavalin A dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown. The number after the sample name indicates the amount of precipitant that was used in crystallization (see Materials and methods and Table 1 ▸).

Figure 8

Diffraction images from crystals of thaumatin dehydrated by adding different amounts of KF13 to the reservoir. The volume of KF13 that was used and the diffraction resolution of the ice rings are shown. The number after the sample name indicates the amount of precipitant that was used in crystallization (see Materials and methods and Table 1 ▸).

Figure 9

Correlation between amounts of KF13 used for crystal drop dehydration and both mosaicity and diffraction resolution of data sets collected for different crystal samples. The green line indicates the minimum value of KF13 volume that caused the ice rings to disappear.