Table 1. The crystallization conditions used to grow the crystals obtained in this study.
The crystals were grown in the thermostatic room at 19 °C using vapor diffusion method of protein crystallization (sitting drop format). The crystallization plates were Cryschem Plate (24 well sitting drop). The volume of crystallization buffer was 0,5 mL in the well of crystallization plate. The mixed volumes of starting drop are listed in table below. The crystallization buffers were homemade using chemicals of highest available purity purchased from Carl Roth GmbH (Karlsruhe, Germany) and Sigma Aldrich (Munich, Germany) and optimized for crystallization of every presented CA isoform.
| Isoform-ligand | Crystallization buffer | PDB ID | Sitting drop |
|---|---|---|---|
| CA II-4 (VD10-49) | 2.03 M sodium malonate (pH 7.5)a | 5LLH | 6 µL of 20 mg mL−1 CA II protein solution and 6 µL of crystallization buffer |
| CA II-13 (VD11-26) | 0.1 M sodium bicine (pH 9) (Roth #9162.3), 0.2 M ammonium sulfate (Sigma-Aldrich #31119) and 2 M sodium malonate (pH 7) | 5LLC | 5 µL of 40 mg mL−1 CA II protein solution and 5 µL of crystallization buffer |
| CA II-2 (VD10-51) | 2.5 M sodium malonate (pH 7.5) and 0.075 M sodium bicine (pH 9) | 5LLE | 6 µL of 60 mg mL−1 CA II protein solution and 6 µL of crystallization buffer |
| CA II-1 (VD12-10) | 0.1 M sodium bicine (pH 9), 0.2 M ammonium sulfate and 2 M sodium malonate (pH 7) | 5LLG | 4 µL of 50 mg mL−1 CA II protein solution and 4 µL of crystallization buffer |
| CA XII-10 (VD10-35) | 0.1 M ammonium citrate (pH 7) (Roth #9488.2), 0.2 M ammonium sulfate and 26% PEG4000 (Roth #0156.1) | 5MSB | 3 µL of 19.7 mg mL−1 CA XII protein solution and 3 µL of crystallization buffer |
| CA XII-16 (VD12-23) | 0.1 M ammonium citrate (pH 7), 0.2 M ammonium sulfate and 26% PEG4000 | 5LLP | 3 µL of 19.7 mg mL−1 CA XII protein solution and 3 µL of crystallization buffer |
| CA XII-12 (VD12-34) | 0.1 M ammonium citrate (pH 7), 0.2 M ammonium sulfate and 26% PEG4000 | 5LLO | 3 µL of 24.4 mg mL−1 CA XII protein solution and 3 µL of crystallization buffer |
Notes.
a
Sodium Malonate solution was prepared by titration of 2.7 M Malonic Acid (“Fluka” #63290) with NaOH powder (“Roth” #9356.2).