Crystallisation was initiated via standard screening in sitting drop 96-well clover-leaf crystallography trays at 18 mg/ml with 3.41 mM AγQK[GGGGG]A in a 1:2 drop ratio of screening agent to protein solution. The trays were incubated at 18 °C. Tetragonal bipyramidal crystals formed within the first 48 hours in 100 mM Bis-tris pH 5.5, 25% (w/v) poly-ethylene glycol 3350 and 200 mM ammonium sulphate.
Crystals were cryo-protected using the above conditions (inclusive of AγQK[GGGGG]A to maintain the ligand:protein complex), and an additional 20% v/v ethylene glycol. Two datasets were collected (Supplementary Table 5 ): a high resolution set at the I03 beamline, Diamond Light Source, Oxford, and a second set on a Rigaku Micromax home source. Data were processed with XiaII/XDS 40 (link). The B-factors for the high-resolution set are higher than expected, but match that of the Wilson B, and the dataset has a normal intensity distribution. An initial model was solved using the existing apo structure 5LEO 20 (link) as a molecular replacement model in PHASER 41 (link), and the corresponding structure autobuilt using PHENIX 42 (link), with the ligand added via visual inspection of the difference map. The structure was updated and refined using COOT 43 (link), PHENIX 42 (link) and PDB-redo 44 (link), resulting in a final structure with an R/Rfree of 19.9%/23.0%.
Crystals were cryo-protected using the above conditions (inclusive of AγQK[GGGGG]A to maintain the ligand:protein complex), and an additional 20% v/v ethylene glycol. Two datasets were collected (