Crystallization

Following the report of Moreau, crystallization was attempted without further purification beyond the nickel column.⁵⁰ Samples of protein at 5–15 mg mL⁻¹ were thawed and screened by sitting drop vapor diffusion. Protein crystals were observed in several conditions of the initial 96-well screens. For example, crystallization was seen in the following conditions of the JCSG⁺ screen (Molecular Dimensions): 1.6 M sodium citrate tribasic dihydrate pH 6.5 (B11); 0.2 M potassium citrate tribasic monohydrate, 20% w/v PEG 3350 (B12); and 0.1 M bicine pH 9.0, 10% w/v PEG 6000 (E10). Follow-up screening gave reproducible crystals near all three conditions. The crystals selected for irradiation were those grown as thin plates from 1.6 M sodium citrate, pH 6.5. Well solution was diluted 1 : 5 with ethylene glycol and brought to a concentration of 50 mM NAD⁺. Crystals were transferred into a 10 μL drop of this mixture at room temperature by cryoloop and soaked for 3 min without back-soaking. The co-crystals were looped and cryocooled by immersion into liquid nitrogen, then directly loaded into pucks for automated handling at the beamline (Advanced Photon Source, Argonne National Laboratory, NE-CAT, 21-ID-D). Crystals were irradiated by 0.979 Å X-rays for 1 s per frame through 180°, and the data processed in XDS. Indexing in the space group P1 gave the best statistics. The crystals diffracted to a resolution of 1.94 Å (Table 1). However, examination of a plot of the Diederichs–Karplus correlation coefficient versus resolution shell showed a plateau beginning at 2.4 Å, suggesting that inclusion of data beyond this resolution would not materially improve the map quality.⁴⁰ This prediction was verified by visual comparison of maps generated by truncating the data set at 2.4 Å to those produced from the full data set. So, even though the mean I/Isigma is greater than 2 in this resolution shell, we felt that truncating the data set here gave the most faithful assessment of its highest resolution.

The model statistics are fairly typical for a structure of this resolution, with the exception of the multiplicity, a consequence of the low symmetry of the space group chosen. The use of the low-symmetry space group means that there are many (eight) copies of the protein in the unit cell, providing an opportunity to compare several different NAD⁺ binding modes.