Crystallization screenings were carried out using the sitting-drop vapor diffusion method and a Mosquito nanoliter-dispensing crystallization robot (TTP Labtech); hits were improved by handmade hanging drops in 24-well plates at the same temperature. Optimized conditions for crystal growth of the different protein complexes (protein concentrations of 20 to 25 mg/ml, molar ratio of 1:1) are as follows: (i) MtPknBL33E-MtGarA: 100 mM Hepes, 4% (w/v) PEG400 (polyethylene glycol, molecular weight 400), 2 M (NH4)2SO4, and 4 mM AMP-PCP (pH 7.5); (ii) MsKGDΔ360-MsGarAΔ44: 50% 2-methyl-2,4-pentanediol (MPD), 100 mM MES, 2 mM ThDP, and 5 mM MgCl2 (pH 6.5); and (iii) MsKGDΔ360,R802A-MsGarAΔ44: 47% MPD, 100 mM Na Hepes, 2 mM ThDP, and 5 mM MgCl2 (pH 7.5). The enamine-ThDP reaction intermediate for KGD in the presence of GarA was obtained by soaking crystals of the MsKGDΔ360,R802A-MsGarAΔ44 (grown as described above) with 20 mM 2-oxoglutarate for 8 min before freezing.

X-ray diffraction data were collected from single crystals at 100 K using synchrotron radiation at beamlines Proxima 1 (Synchrotron Soleil, Saint-Aubin, France) and ID14-1 (ESRF, Grenoble, France). Data processing was carried out with programs XDS (58) and AIMLESS (59).

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