4.3. Crystallisation

The concentrated protein was mixed with acarbose in a molar ration of 4:1 before the initial screening in 96 well format using commercially available screens. An initial hit (0.2 M NaCl, 0.1 M Na-acetate pH 4.6, 30% MPD) was further refined in 24-well format using the initial crystals as seeds. Crystals suitable for data collection were cryoprotected using 25% glycerol and flash frozen in liquid nitrogen prior data collection.

Prior to providing the sample to York, the protein was deglycosylated using Endo-H treatment. The protein was concentrated using Amicon (Merck, Germany) filter units and stored at −80 °C for later use. For the crystallization, the protein was mixed with 5 mM acarbose prior to setting up the screen. Initial screens were set up in a 96-well sitting drop format using commercially available screens. Initial hits were further refined in a 24-well hanging drop format. The best crystals grew in 0.1 M di-hydrogen phosphate, 1.8 M ammonium sulphate. Crystals were cryoprotected by addition of ethylene glycol to a final concentration of 15%. The crystals were flash frozen in liquid nitrogen prior to data collection.

Prior to crystallization, the protein was concentrated to 22.5 mg/mL by ultrafiltration in an Amicon centrifugation filter unit (Millipore), aliquoted to 50 µL; aliquots that were not immediately set up for crystallization were flash frozen in liquid nitrogen and stored at ™80°C to use later in optimizations. Initial crystallization experiments were carried out in the presence or absence of 4 mM CaCl₂ and 40 mM acarbose. An initial hit was obtained for an acarbose complex, in just one condition (H3, Bis-tris 5.5, 25% w/v PEG3350) of JCSG screen (Figure 6a), out of total 192 conditions in two initial screens set up – JCSG and PACT premier™ HT-96 (Molecular Dimensions (Suffolk, UK)). The crystals were imperfect and were used to make the seeding stock. The seeding stock was prepared and microseed matrix screening (MMS, recent review in [14]) carried out using an Oryx robot (Douglas Instruments (Hungerford, UK)) according to the published protocols [36,37]. Briefly, crystals were crushed, and diluted with ~50 µL of mother liquor. The solution was transferred into a seed bead containing reaction tube and vortexed for three minutes. The seeding stock was used straightaway, and the remaining seeds were frozen and kept at ™20 °C. MMS was carried out in the PACT screen, giving a significant number of hits (Figure 6b). Crystals from condition A11 were used to make a seeding stock for the next seeding round. This time it was not a “classical” MMS-seeding into a random screen, but rather seeding into an optimization screen based on the initial conditions, but with different pH, salts and PEGs/PEG concentrations. The crystallization drops contained 150 nl protein + 50 nl seeding stock + 100 nl mother liquor from a new random screen. The final, good quality crystal was obtained in 12% PEG 3350 0.2 M NaNO₃, CAPS pH 11.0 (Figure 6c).

Crystal optimization using microseed matrix screening.