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

STAR Protoc. 2022-01;

Fatma Betul Ertem, Omur Guven, Cengizhan Buyukdag, Oktay Gocenler, Esra Ayan, Busra Yuksel, Mehmet Gul, Gozde Usta, Bar?s Cak?lkaya, J Austin Johnson, E Han Dao, Zhen Su, Frederic Poitevin, Chun Hong Yoon, Christopher Kupitz, Brandon Hayes, Mengning Liang, Mark S Hunter, Alexander Batyuk, Raymond G Sierra, Gihan Ketawala, Sabine Botha, ?a?da? Da?, Hasan DeMirci

Products/Services Used	Details	Operation
Proteins, Expression, Isolation and Analysis	… Ni-NTA Agarose resin, QIAGEN, Cat#30250. PreScission, GenScript, Cat#Z02799. Terasaki Plate, Greiner Bio-One, Cat#654 102?… pET28a(+) Construct, GenScript, Software and algorithms?…	Get A Quote

摘要

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).