Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus.
The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4(1)32 or P4(3)32, with unit-cell parameters a = b = c = 166.8 angstroms. Diffraction data were collected to a maximum resolution of 2.7 angstroms.
Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d'Ingénieurs de Luminy-Case 925, 13288 Marseille CEDEX 9, France.
Stéfano
S
Ricagno
Bruno
B
Coutard
Sacha
S
Grisel
Nicolas
N
Brémond
Karen
K
Dalle
Fabienne
F
Tocque
Valérie
V
Campanacci
Julie
J
Lichière
Violaine
V
Lantez
Claire
C
Debarnot
Christian
C
Cambillau
Bruno
B
Canard
Marie Pierre
MP
Egloff
D003001:Cloning, Molecular; D003460:Crystallization; D004722:Endoribonucleases; D004926:Escherichia coli; D046911:Macromolecular Substances / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D004722:Endoribonucleases; C000657925:nidoviral uridylate-specific endoribonuclease