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Deducing the Crystal Structure of MERS-CoV Helicase.

Identifieur interne : 000849 ( PubMed/Corpus ); précédent : 000848; suivant : 000850

Deducing the Crystal Structure of MERS-CoV Helicase.

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Methods in molecular biology (Clifton, N.J.) [ 1940-6029 ] ; 2020.

RBID : pubmed:31883088

Abstract

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.

DOI: 10.1007/978-1-0716-0211-9_6
PubMed: 31883088

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<author><name sortKey="Cui, Sheng" sort="Cui, Sheng" uniqKey="Cui S" first="Sheng" last="Cui">Sheng Cui</name>
<affiliation><nlm:affiliation>NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China. Cui.sheng@ipb.pumc.edu.cn.</nlm:affiliation>
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<author><name sortKey="Hao, Wei" sort="Hao, Wei" uniqKey="Hao W" first="Wei" last="Hao">Wei Hao</name>
<affiliation><nlm:affiliation>NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China.</nlm:affiliation>
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<front><div type="abstract" xml:lang="en">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.</div>
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<Abstract><AbstractText>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.</AbstractText>
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<AffiliationInfo><Affiliation>NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P. R. China. Cui.sheng@ipb.pumc.edu.cn.</Affiliation>
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<KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">Coronavirus</Keyword>
<Keyword MajorTopicYN="Y">Crystallization</Keyword>
<Keyword MajorTopicYN="Y">Helicase</Keyword>
<Keyword MajorTopicYN="Y">Structure determination</Keyword>
<Keyword MajorTopicYN="Y">nsp13</Keyword>
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Deducing the Crystal Structure of MERS-CoV Helicase.

Deducing the Crystal Structure of MERS-CoV Helicase.

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Abstract

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