Glutamine synthetase from Methanothermococcus thermolithotrophicus at a resolution of 2.43 A

Differences in the regulation mechanisms of the glutamine synthetase from methanogenic archaea unveiled by structural investigations.

Glutamine synthetase from Methanothermococcus thermolithotrophicus

Methanothermococcus thermolithotrophicus DSM 2095

Protein was crystallized fresh without any freezing step by using the sitting drop method on a 96-Well MRC 2-Drop Crystallization Plates in polystyrene (SWISSCI, United Kingdom) under anaerobic conditions (N2:H2, gas ratio of 97:3). The enzyme was crystallized at 15 mg/ml in 25 mM Tris/HCl pH 7.6, 10% v/v glycerol, 150 mM NaCl, and 2 mM dithiothreitol. The crystallization reservoir contained 90 uL of mother liquor (20 % w/v polyethylene glycol 3,350 and 100 mM potassium sodium tartrate), the crystallization drop contained a mixture of 0.55 uL protein and 0.55 uL mother liquor. Crystals were soaked in the mother liquor supplemented with 25% v/v ethylene glycol prior to freezing in liquid nitrogen.

The structure was refined by applying translation libration screw model (TLS) and adding riding hydrogens. In addition, this dataset was refined by applying the following twin operator: 1/2h+1/2k,3/2h-1/2k,-l. Hydrogens were omitted in the final deposited model.

Nitrogen-assimilation, methanogenic archaea, allosteric activation, hydrogenotrophic, thermophile, marine, glutamate, ATP, LIGASE