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![[Figure 2]](cy5023fig2mag.jpg)
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Figure 2
Dehydration methods. Method 1: serial transfer of crystals from the crystallization drop to 50 µl drops containing increasing amounts of dehydrating solution. The dehydrating solution can consist of mother liquor with increasing precipitant concentration or it can be supplemented with increasing concentrations of cryoprotective agents such as PEG 400, glycerol or MPD. Depending on the stability of the crystal, the concentration of dehydrating agent can be increased in steps of 5% up to ∼30%(w/v), steps of 0.5% up to∼5%(w/v) or as follows: 1, 2, 3, 4, 5, 10, 15, 20%. Soaking time can also vary from 5 to 15 min (in this case crystals are air dehydrated) to days (in this case dehydrating drops are equilibrated against a reservoir containing dehydrating solution) (Schick & Jurnak, 1994  ; Esnouf et al., 1998). Method 2: add dehydrating solution slowly to the drop containing the crystal (about eight times the crystallization drop volume) and air dehydrate for more than 30 min. Dehydrating solution consists of crystallizing conditions with a 10–12% increase in precipitant concentration (e.g. PEG, MPEG) and ∼5–10% addition of cryoprotective agent such as glycerol (Haebel et al., 2001). Method 3: transfer the crystal from the crystallization drop into a 5 µl hanging drop of dehydrating solution and equilibrate against a reservoir with the same dehydrating solution (dehydrating solution: crystallization condition containing 5–10% more precipitating agent; incubation time: 12–16 h; Heras et al., 2003). Method 4: After crystal growth, equilibrate the crystallization drop against reservoirs containing increasing concentration of dehydrating agent (dehydrating solution: mother liquor containing increasing concentrations of either precipitant or low-molecular-weight PEG, glycerol or MPD. Concentration is increased in steps of 5%. Incubation time: 8–12 h each). For very fragile crystals it is recommended that these dehydration procedures be performed at 277 K. Crystal soaking without dehydration is performed similarly to method 1 by, for example, transferring the crystal to 10 µl of soaking solution consisting of mother liquor containing a cryoprotectant (∼20% glycerol or 40–50% malonate) and incubating for a few seconds to 5 min (Holyoak et al., 2003). |
![[Figure 2]](cy5023fig2.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
