Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography

• Published in: STAR protocols Vol. 3; no. 1; p. 101158
• Main Authors: Ertem, Fatma Betul, Guven, Omur, Buyukdag, Cengizhan, Gocenler, Oktay, Ayan, Esra, Yuksel, Busra, Gul, Mehmet, Usta, Gozde, Cakılkaya, Barıs, Johnson, J. Austin, Dao, E. Han, Su, Zhen, Poitevin, Frederic, Yoon, Chun Hong, Kupitz, Christopher, Hayes, Brandon, Liang, Mengning, Hunter, Mark S., Batyuk, Alexander, Sierra, Raymond G., Ketawala, Gihan, Botha, Sabine, Dağ, Çağdaş, DeMirci, Hasan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.03.2022; Elsevier
• Subjects: Coronavirus 3C Proteases - chemistry; Coronavirus 3C Proteases - genetics; Crystallization; Crystallography, X-Ray; Humans; Models, Molecular; Protein Biochemistry; Protein expression and purification; Protocol; SARS-CoV-2 - enzymology; Structural Biology; X-ray Crystallography; Protein Biochemistry; X-ray Crystallography; Protein expression and purification; Structural Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101158

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). [Display omitted] •Protocol for SARS-CoV-2 Main protease production and purification•Sitting drop, microbatch screening (under oil) approach for protein crystallization•Data collection and structure determination procedure for SARS-CoV-Mpro 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.