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Materials and methods
Crystallization. CEAS purification and initial crystallization was carried out as previously described [5]. Crystals of two morphologies were grown by the hanging-drop vapour Lomeguatrib technique using 1.6 M ammonium sulfate as precipitant, in the presence of 0.1 M HEPES, pH 7.4, and a threefold molar excess of ThDP. Crystals used for soaking experiments were of the rod morphology and belonged to the space group P212121. The asymmetric unit contained four CEAS subunits (labelled A, B, C and D) arranged as a dimer of dimers, with the active sites at the monomer interfaces of each dimer [5].
Precipitant exchange was accomplished by soaking the rod-shaped crystals for 24 h in 2 M dipotassium l(+)-tartrate and 0.1 M HEPES, pH 7.5. Following precipitant exchange, soaking experiments were carried out in 2 M dipotassium l(+)-tartrate and 0.1 M HEPES, pH 7.5, containing either dl-G3P at 3 mM, or both dl-G3P and GVA at 3 and 10 mM, respectively. Crystals were soaked for 12 h before cryoprotection and subsequent flash-cooling by plunging into liquid nitrogen. Cryoprotection comprised sequential soaking of crystals for 30 s in solutions of 2 M dipotassium l(+)-tartrate and 0.1 M HEPES, pH 7.5, containing 10% (v/v) and then 25% (v/v) glycerol. All crystallization, precipitant exchange and substrate soaking experiments were carried out at 17 °C.