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This N-terminal insertion drives conformational changes in the TMD, including the outward movement of transmembrane helix 6 (TM6) and reorganization of TM5 and TM7, that create a binding surface for heterotrimeric G proteins on the intracellular face of the receptor

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To evaluate growth on solid media, in all cases -with the exception of the metE mutant, which only grew in the presence of B12 and, therefore, plating with and without B12 was performed from liquid cultures supplemented with B12- the liquid cultures grown without B12 were plated in solid 7H10-ADC plates supplemented with or without AdoCbl (10 g/mL) and/or L-Met (25 g/mL)

Briefly, proteins were buffer-exchanged after IMAC into 20 mM TrisHCl pH 7.5, 250 mM sodium chloride using PD-10 columns (GE Healthcare) at room temperature