Crystal screen ht

The purified un-tagged MmATD was screened for crystallization conditions using different screens (Index, Crystal Screen HT, PEG/Ion and PEGRx from Hampton Research, USA) at two different temperatures—4 °C and 20 °C. Mosquito Crystal (TTP LabTech, UK) crystallization robot was used to set up crystallization experiments using sitting-drop vapor diffusion method by mixing 1 µl protein and 1 µl reservoir buffer in a 96-well MRC plate with three sub-wells (Molecular Dimensions, UK). The initial hits from the screens were further expanded for optimization using sitting-drop vapor diffusion method in 96-well format MRC plates having three sub-wells. Reservoir buffer with 0.1 M Bicine (pH 8.0) and 15% PEG1500 yielded good diffraction-quality crystals.

TfAA10A crystals were initially obtained using sitting drop vapor diffusion and a 96-well plate with Crystal Screen HT from Hampton Research (Aliso Viejo, CA). Reservoirs contained 50 µL of well solution and drops had 0.2 µL of well solution and 0.2 µL of protein solution. A Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA) was used for setting up the screens. The best crystals were grown at 20 °C with 0.1 M Sodium acetate trihydrate pH 4.6, 20% v/v 2-Propanol and 0.2 M Calcium chloride dihydrate as the well solution. The protein solution that was used for crystallization contained 8.5 mg/mL of protein in 20 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol and 5% ethylene glycol.

L-AucA and D-AucA were dissolved in water to a final concentration of 80 mg/mL. The peptide solutions were mixed 1:1 to yield an 80 mg/mL racemate of D- and L-AucA which was diluted two-fold with water to yield 40 mg/mL DL-AucA. Both 80 mg/mL and 40 mg/mL racemate concentrations were subject to sparse-matrix crystallization screening using Crystal Screen HT (HR2-130) and SaltRx HT (HR2-136) from Hampton research. 50 μL of each precipitant condition solution was added into the wells of a SWISSCI 96-well plate. The two racemate concentrations were each mixed 1:1 with the precipitant in a 0.4 μL sitting drop, yielding 384 crystallization drops across two screens. The best conditions which produced single, three-dimensional crystals were selected for optimization to produce crystals suitable for X-ray diffraction. LnqQ was crystallized in the same manner, using 27 mg/mL and 13.5 mg/mL of racemate, respectively. Details of crystallization conditions, X-ray diffraction data collection, and structure solution and refinement can be found in the Supplementary Methods. Data refinement statistics are given in the Supplementary Information Table S5 and the refined models of racemic AucA and LnqQ have been deposited in the Protein Data Bank with the PDB codes 8AVR, 8AVS, 8AVU, 8AVT and 7P5R (Supplementary Data 1–5).