Compositions of hepatitis C virus NS5B polymerase and methods for crystallizing same
OwnerCharles A. Lesburg, Michael Cable +3inventors
InventorsCharles A. Lesburg, Michael Cable, Zhi Hong, Anthony F. Mannarino, Patricia C. Weber
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Abstract
The invention relates to the purification, crystallization of and structure of hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. Also, crystallization conditions for NS5B are provided. Further, the atomic coordinates for the NS5B protein are disclosed. Examples of its use for the determination of the three-dimensional atomic structures of HCV NS5B or HCV NS5B in complex with substrates or substrate analogs or inhibitors are also provided.
Claims23
A crystalline composition comprising a HCV NS5B polypeptide.
A composition comprising a NS5B protein having at least one amino acid substitution at position 335, 344 or 550 of SEQ ID NO:
- 1.
A composition comprising a NS5B protein having at least one amino acid substitution at position 335, 344 or 550 of a histidine-tagged NS5B protein.
A method of preparing purified, crystalline HCV NS5B comprising the steps of:
- (a) stabilizing a NS5B protein preparation in a solution containing a protein stabilizing agent;
- (b) subjecting the NS5B protein preparation to cation exchange chromatography in a buffer containing glycine;
- and
- (c) allowing crystals to form in a precipitant containing a protein stabilizing agent, a salt and polyethylene glycol under conditions in which crystallization occurs.
A machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of a NS5B polypeptide according to Table 1, or a homologue thereof wherein said homologue comprises alpha carbon atoms that have a root mean square deviation between equivalent alpha carbon atoms of the polypeptide of not more than 3.0 Å.
A method of obtaining structural information concerning a molecule of unknown structure by using the structure coordinates set forth in Table 1, comprising the steps of:
- (a) generating x-ray diffraction data from said crystallized molecule;
- and
- (b) applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 1 to said x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule.
A method of obtaining structural information concerning a molecular complex of unknown structure by using the structure coordinates set forth in Table 1, comprising the steps of:
- (a) generating x-ray diffraction data from said crystallized molecule;
- and
- (b) applying crystallographic phases derived from at least a portion of the structure coordinates set forth in Table 1 to said x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule.
Dependencies are inferred from the claim text.