For complexes containing AMPCPP/GMPCPP, Pol II was co-crystallized with nucleic acids in the presence of 8 mM magnesium chloride and were soaked with NTP ligand by including 2 mM AMPCPP/GMPCPP in all cryo protectant solutions.
For derivatization experiments, crystals were incubated for 24 h in the respective cryo-protectant solution saturated with the ligand and flash-frozen in liquid nitrogen.
The crystals were soaked in cryo-protectant solution composed of 35% (w/v) poly ethylene glycol) 4000, 2 or 20 mM A/S, 50 mM myo-inositol, and 100 mM sodium acetate buffer (pH 4.6) and flash-frozen in a nitrogen stream at 100 K.
The well-developed crystals were safely harvested from the tiny droplet into a large well with so-called cryo-protectant solution.
Prior to the data collection, the SmCl3 soaked single crystal was transferred to the cryo-protectant solution (mother liquid supplemented with 20% glycerol and 1 mM SmCl3), and then frozen at 100 K in a nitrogen gas cold stream (Oxford Cryosystems, Oxford, UK).
Where needed, heme ligands were added into the cryo-protectant solution.
Data collection and processing: Single crystals were flash cooled in liquid nitrogen (cryo-protectant solution contained NaOAc (0.1 m, pH 4.8), NaCl (11.3 %, w/ v) and glycerol (15 %)).
Crystals were transferred to a cryo-protectant solution of (88% saturated Li2SO4, 14 mM magnesium formate, 20 mM Tris HCl (pH 8.0), and 0.1% (v/v) βME) by gradually changing the drop solution in 20 min, followed by cooling in liquid nitrogen.
The H3.5 nucleosome crystals were soaked for 5 10 s at room temperature in a cryo-protectant solution, containing 20 mM potassium cacodylate (pH 6.0), 40 mM KCl, 70 mM MnCl2, 29%% 2-methyl-2,4-pentanediol, and 2 % trehalose, and were flash-cooled in a stream of N2 gas (100 K).
