Structural genomics of the SARS coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the Nsp9 protein.
The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses. SARS-CoV is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent. A SARS-CoV whole-genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains. These studies are expected to greatly facilitate drug design. The genomes of coronaviruses are between 27 and 31.5 kbp in length, the largest of the known RNA viruses, and encode 20-30 mature proteins. The functions of many of these polypeptides, including the Nsp9-Nsp10 replicase-cleavage products, are still unknown. Here, the cloning, Escherichia coli expression, purification and crystallization of the SARS-CoV Nsp9 protein, the first SARS-CoV protein to be crystallized, are reported. Nsp9 crystals diffract to 2.8 A resolution and belong to space group P6(1/5)22, with unit-cell parameters a = b = 89.7, c = 136.7 A. With two molecules in the asymmetric unit, the solvent content is 60% (V(M) = 3.1 A(3) Da(-1)).
Campanacci
Valérie
V
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20, France.
Egloff
Marie-Pierre
MP
Longhi
Sonia
S
Ferron
François
F
Rancurel
Corinne
C
Salomoni
Aurelia
A
Durousseau
Cécile
C
Tocque
Fabienne
F
Brémond
Nicolas
N
Dobbe
Jessika C
JC
Snijder
Eric J
EJ
Canard
Bruno
B
Cambillau
Christian
C
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D023281:Genomics; D016601:RNA-Binding Proteins / Q000737:chemistry*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry*
D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C087633:nonstructural protein, coronavirus; C483945:nsp9 protein, SARS virus