Protein Crystallization

CTX-M-14 E166A (20 mg/ml) was crystallized in 1.0 M potassium phosphate pH 7.9 at 20° C using hanging drops in a 1:1 ratio (protein:buffer). Single crystals formed in 3–4 days. 10 mM compound 1 dissolved in the crystallization buffer was then added to the crystal drops and allowed to soak into the crystals. Soaking times were as follows: 24 hrs for the 1.30 Å E166A structure; 4 days for the 1.18 Å S70G structure; 5 days for the 0.85 Å E166A structure. After soaking, the crystals were collected and cryo-cooled using a solution of the crystallization buffer with 30% sucrose added.