All crystallization trials were performed using the sitting-drop vapor diffusion method at 296 K. The crystals were soaked in cryoprotectant by incrementally increasing the glycerol concentration up to 20%. The crystals were then mounted on cryo-loops (Hampton Research Corp., Aliso Viejo, CA, USA), flash-cooled, and stored in liquid nitrogen.

For A3B(L65Y)3 without nucleotides, crystals of nucleotide-free A3B3 [eA3B(L65Y)3] [10 mg/ml in 20 mM tris-HCl (pH 7.5), 100 mM NaCl, and 10% (v/v) glycerol] were grown at 23°C by vapor diffusion after an equal volume (0.3 μl) was mixed with 0.1 M NaCl, 0.1 M ammonium acetate, 0.1 M bis-tris propane (pH 6.5), and 28% (w/v) PEG-3350 (polyethylene glycol, molecular weight 3350).

For A3B(L65Y)3 with AMP-PNP, crystals of AMP-PNP bound A3B3 [bA3B(L65Y)3] [12.1 mg/ml in 20 mM tris-HCl (pH 7.5), 100 mM NaCl, 10%(v/v) glycerol incubated with addition of 120 mM MES (pH 6.0), 6 mM AMP-PNP, and 6 mM MgCl2 for 1 hour on ice] were grown at 23°C by vapor diffusion after an equal volume (0.5 μl) was mixed with 0.2 M NaCl, 0.1 M MES (pH 5.5), and 27% (w/v) PEG-3350.

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