Bovine calf serum (bcs)

Crystallization conditions for the BlaC G132N and K234R mutants were screened by sitting-drop vapor diffusion using the BCS, Morpheus, JCSG+, and PACT premier (Molecular Dimensions) screens at 293 K with 100-nl drops at a 1:1 ratio (51 (link)). The plates were pipetted by using the NT8 drop setter (Formulatrix). BlaC K234R was used at concentrations of 8 mg ml⁻¹ and 10 mg ml⁻¹ in 100 mM morpholineethanesulfonic acid (MES)-NaOH buffer (pH 6.4). Multiple conditions across the screens showed growth on microneedles or multilayer plates, which were used for seeding and further buffer optimization. However, that did not yield any usable crystals. BlaC G132N was used at a concentration of 18 mg ml⁻¹ in 100 mM MES-NaOH buffer (pH 6.4). To obtain crystals, it was necessary to supplement the protein with 100 mM sodium phosphate buffer and cross-seed with crystals from another BlaC mutant. A crystal of BlaC G132N grew within 2 months in 0.1 M Morpheus buffer 1 (pH 6.5) with 0.09 M halogens and 30% (wt/vol) ethylene glycol polyethylene glycol 8000 as the precipitant. The crystal was mounted on a cryoloop in mother liquor with additional 20% glycerol and vitrified in liquid nitrogen for data collection.

Human NEMO capped peptide (residues 226-235, acetyl-KLAQLQVAYH-amide, synthesized by Thermo Scientific) was dissolved to 2 mM in peptide buffer (20 mM HEPES, 150 mM NaCl, pH 7.2) and DMSO (5.67%). Peptides were then added to 3CLpro C145S at a 20-fold molar excess, before overnight incubation at 4 °C. The mixture was centrifuged to remove precipitants, and the supernatant was concentrated to 6.5 mg/mL for crystallization screens.
TOP96 (Rigaku Reagents, Japan), BCS (Molecular Dimensions, UK) and GRAS2 (Hampton Research, USA) screens were run using a Gryphon (Art Robbins Instruments, USA). 0.2 µL of protein solution at 6.5 mg/mL was added to 0.2 µL of crystallization matrix in a sitting-drop vapor diffusion setup at 18C. GRAS2 screen condition F11 (0.1 M Sodium phosphate dibasic dihydrate, pH 9.3, 10 mM Calcium chloride, 20% w/v Polyethylene glycol 3,350), yielded platelike single crystals. These crystals were isolated, cryoprotected by supplementation with 20% glycerol, mounted into ALS style, 0.05-0.1 mm loops (Hampton Research, USA) and flash frozen in liquid nitrogen.