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Figure 2
The use of microseeds improves apo Sirt3 crystallization. (a) Representative initial apo Sirt3 crystals obtained in a solution consisting of 60%(v/v) Tacsimate, 0.2 M lithium sulfate at 4°C after a week. (b) UV image of (a). (c) Schematic representation of crystal formation in the Index screen (Hampton Research) using MMS with two different crystallization-drop compositions at two different temperatures. A red-coloured circle indicates crystal formation in both drops, while an orange circle indicates crystal formation in only one drop. A colourless condition represents no crystal formation. Conditions containing high salt concentrations are marked in light grey and crystallization conditions with more than 10%(w/v) PEG are marked in light blue. Apo Sirt3 crystallizes in mainly PEG 3350-containing conditions preferentially under acidic conditions at 4°C as well as 20°C. (d) Representative crystals of apo Sirt3 obtained with microseeds in Index screen condition D11 [0.1 M bis-tris buffer, 28%(w/v) PEG MME 2000 pH 6.5] at 4°C. (e) UV image of (d). (f) Representative apo Sirt3 crystals obtained with microseeds in Index screen condition G6 [0.2 M ammonium acetate, 0.1 M bis-tris buffer, 25%(w/v) PEG 3350 pH 5.5]. (g) UV image of (f). (h) Superposition of apo Sirt3 (PDB entry 3gls, chain A, light grey) with the improved apo Sirt3 structure (chain A, slate blue). Both structures feature the two-domain structure typical of sirtuins and are very similar (r.m.s.d. of 0.25 Å for all Cα atoms).

ISSN: 2053-230X
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