Purification, crystallization, and preliminary X-ray analysis of L-A: A dsRNA yeast virus

The L-A virus (LAV) particle is a specialized compartment for the transcrip tion and replication of double-stranded RNA. It is 390 Angstrom in diameter and infects yeast. The particle is formed by a capsid containing 120 copie s of a 680-residue gene product arranged with T = I icosahedral symmetry, a pproximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb lin ear, duplex RNA. LAV crystals diffracting to at least 4.5-Angstrom resoluti on were grown in a combination of polyethylene glycol 8000, ethylene glycol , and lithium chloride. Following crystallization the reservoir solution wa s replaced by a 2 X concentrated reservoir solution in order for ethylene g lycol to function as a cryoprotectant even though initial crystals would no t grow at sufficiently high concentrations of ethylene glycol for cryoprote ction. A complete data set was collected to 6-Angstrom resolution from a fr ozen crystal obtained with this procedure. The crystals belong to space gro up P2(1). The unit cell dimensions are a = 406.7 Angstrom, b = 403.3 Angstr om, c = 572.5 Angstrom, beta = 90.3 degrees with two virus particles in the unit cell. The particle orientation was determined with the rotation funct ion and the particle center was estimated on the basis of packing considera tions. (C) 2001 Academic Press.