ABSTRACT
The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).
Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/genetics , Crystallization , Crystallography, X-Ray , HumansABSTRACT
The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.
Subject(s)
COVID-19 Drug Treatment , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Repositioning , SARS-CoV-2 , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Dimerization , Molecular Conformation , Molecular Docking Simulation , Principal Component Analysis , Protein Conformation , Recombinant Proteins/chemistry , TemperatureABSTRACT
OBJECTIVE: To compare the effectiveness of different mask types in limiting the dispersal of coughed air. METHOD: The Schlieren method with a single curved mirror was used in this study. Coughed air has a slightly higher temperature than ambient air, which generates a refractive index gradient. A curved mirror with a radius of curvature of 10 m and a diameter of 60 cm was used. The spread of the cough wavefront was investigated among five subjects wearing: (1) no mask; (2) a single surgical mask; (3) a double surgical mask; (4) a cloth mask; (5) a valveless N95 mask; and (6) a valved N95 mask. RESULTS: All mask types reduced the size of the contaminated region significantly. The percentage reduction in the cross-sectional area of the contaminated region for the same mask types on different subjects revealed by normalized data suggests that the fit of a mask plays an important role. CONCLUSIONS: No significant difference in the spread of coughed air was found between the use of a single surgical mask or a double surgical mask. Cloth masks may be effective, depending on the quality of the cloth. Valved N95 masks exclusively protect the user. The fit of a mask is an important factor to minimize the contaminated region.