Crystal preparation

For X-ray crystallography, Gdx-Clo and monobody Clo-L10 were purified as described above. For the Clo purification, the size exclusion buffer contained 200 mM NaCl, 10 mM HEPES pH 8.1, and 10 mM Gdm⁺ or 20 mM phenylGdm⁺. Proteins were concentrated to 10 mg/ml, Clo-L10 was supplemented with 4 mM DM, and monobody and Gdx-Clo dimer were mixed in a 2.1:1 ratio. The protein solution was then mixed with an equal volume of crystallization solution (0.3 μl in 96-well plates). Initial hits grew in 200 mM CaCl₂, 0.1 M Tris/HCl pH 8.0 and 32.5% PEG 600. Crystals were subsequently improved by addition of charged detergents lauryldimethylamine-N-Oxide (LDAO; final concentration 6.6 mM), dimethyldodecylphosphine oxide (Apo12; final concentration 2 mM), or octylGdm⁺ (final concentration 3.3 mM) to the protein solution prior to admixture with the crystallization solution (0.45 μl protein/detergent mixture together with 0.3 μl crystallization solution). Optimized crystals typically grew to their maximum size in 14 days in a wide range of salt and pH conditions with 32−36% PEG 600. For selenomethionine-incorporated protein, the best diffracting crystals were obtained with Apo12 supplementation, and crystallization solution 0.1 M LiNO₃, 0.1 M N-(2-Acetamido)iminodiacetic acid (ADA) pH 6.8, and 35% PEG 600. For phenylGdm⁺ bound protein, the best diffracting crystals were obtained with Apo12 supplementation, and crystallization solution 0.1 M LiNaSO₄, 100 mM Tris pH 8.8 and 34% PEG 600. For the octylGdm⁺ bound structure, octylGdm⁺ was used as the detergent additive, and crystallization solution contained 0.1 M calcium acetate, 0.1 M HEPES pH 7.5 and 33% PEG 600. Crystals were frozen in liquid nitrogen before data collection at the Life Sciences Collaborative Access Team beamline 21-ID-D at the Advanced Photon Source, Argonne National Laboratory.