Deducing the Crystal Structure of MERS-CoV Helicase #MMPMID31883088
Cui S; Hao W
Methods Mol Biol 2020[]; 2099 (ä): 69-85 PMID31883088show ga
RNA virus encodes a helicase essential for viral RNA transcription and replication when the genome size is larger than 7 kb. Coronavirus (CoV) has an exceptionally large RNA genome (~30 kb) and it encodes an essential replicase, the nonstructural protein 13 (nsp13), a member of superfamily 1 helicases. Nsp13 is among the evolutionary most conserved proteins not only in CoVs but also in nidovirales. Thus, it is considered as an important drug target. However, the high-resolution structure of CoV nsp13 remained unavailable even until more than a decade after the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, which hindered the structure-based drug design. This is in part due to the intrinsic flexibility of nsp13. Here, we describe protocols of deducing the crystal structure of Middle East respiratory syndrome coronavirus (MERS-CoV) helicase in detail, which include protein expression, purification, crystallization, enzymatic characterization, and structure determination. With these methods, catalytically active recombinant MERS-CoV nsp13 protein can be prepared and crystallized and the crystal structure can be solved.
|Coronavirus Infections/*virology[MESH]
|Crystallization[MESH]
|Humans[MESH]
|Methyltransferases/*chemistry[MESH]
|Middle East Respiratory Syndrome Coronavirus/*enzymology[MESH]
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Methods+Mol+Biol 2020 ; 2099 (ä): 69-85
