2.1. Protein production and crystallization  

The expression, purification and crystallization of hydrogenated H-RAS, residues 1–166 (referred to here as Ras), has previously been reported (Knihtila et al., 2015 ▸). Briefly, large crystals of hydrogenated Ras were grown in sitting drops with volumes of between 50 and 100 µl set up in nine-well glass plates. The drops contained a 1:1 ratio of protein solution and reservoir solution. The reservoir solution was a mixture of 25 ml Hampton Research PEG Screen condition No. 28 [200 mM calcium acetate, 20%(w/v) PEG 3350, 0.1%(w/v) n-octyl-β-d-glucoside] and 7.5 ml protein stabilization buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT). Once the crystals had stopped growing, the hydrogenated reservoir solution was replaced with an identical reservoir solution prepared with D₂O. The pD of the crystallization solution used for the exchange by vapor diffusion was 8.4. After two months of exchange, seven crystals were mounted in a thin-walled quartz capillary with a plug of deuterated solution at pD 8.4 (Hampton Research).