SARS-CoV-2 Mpro was diluted to 5 mg/ml and incubated with 1.5 mM inhibitor at 4°C overnight. Samples were centrifuged at 13,000g for 1 min to remove precipitate. Crystals were grown by mixing the protein-inhibitor sample with an equal volume of crystallization buffer [20% PEG 3000 (polyethylene glycol, molecular weight 3000), 0.2 M Na citrate (pH 5.6)] in a vapor diffusion, hanging drop apparatus. A cryoprotectant solution of 35% PEG 3000 and 30% glycerol was added directly to the drop and soaked for 15 min. Crystals were then flash-frozen in liquid nitrogen for x-ray diffraction.
X-ray diffraction data for the SARS-CoV-2 Mpro structures were collected on the Structural Biology Center 19-ID beamline at the Advanced Photon Source in Argonne, IL and processed with the HKL3000 software suite (55 (link)). The CCP4 version of MOLREP was used for molecular replacement using a previously solved SARS-CoV-2 Mpro structure, PDB ID: 7BRR as a reference model for the dimeric P21 Mpro withUAWJ246 (56 (link)). PDB ID: 6YB7 was used as the reference model for the C2 monomeric Mpro with calpain inhibitors II and XII and UAWJ247 , and the P1 dimeric structure with UAWJ248 . PDB ID: 6WTT was used as the reference model for the P3221 trimer with UAWJ246 . Rigid and restrained refinements were performed using REFMAC, and model building was performed with COOT (57 (link), 58 (link)). Protein structure figures were made using PyMOL (Schrödinger LLC).
X-ray diffraction data for the SARS-CoV-2 Mpro structures were collected on the Structural Biology Center 19-ID beamline at the Advanced Photon Source in Argonne, IL and processed with the HKL3000 software suite (55 (link)). The CCP4 version of MOLREP was used for molecular replacement using a previously solved SARS-CoV-2 Mpro structure, PDB ID: 7BRR as a reference model for the dimeric P21 Mpro with
