1Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
2Department of Biochemistry, Indian Institute of Science, Bangalore.
Earlier determination of the high-resolution three-dimensional structure of Physalis mottle virus (PhMV) using screen-less oscillation data collected on films suffered from excessive radiation damage. Therefore, structure redetermination using data collected from an imaging plate were initiated. PhMV was propagated on Nicotiana glutinosa and purified from infected leaves by differential centrifugation. Empty capsids formed in vivo (top component), were separated from the particle containing RNA (bottom component) by sucrose density gradient ultracentrifugation. Crystallization condition was optimized for the micro batch method under oil by modification of earlier conditions standardized for crystallization in depression slides. Crystals were obtained using a virus concentration of 10–20 mg/ml in the cocktail containing 0.2mM sodium acetate, pH5.6, 2mM CaCl2, 2mM DTT, 2.5–3.0% (w/v) polyethylene glycol 8000 and 10% glycerol. X-ray diffraction data were collected at 100K from a single crystal using X-rays from a rotating anode X-ray generator (CuKá) equipped with a MARRESEARCH imaging plate detector. Bottom component crystals were found to belong to space group P1 (unit-cell parameters a=285.40 Å, b=288.36 Å, c=290.61Å, alpha=62.92°, beta=65.30 ° and gamma=60.85 °) in contrast to the earlier crystal form of trigonal R3. The data consisting of 80.2% of the theoretically possible reflections to 3.4 Å had an R-merge of 14.7%. The unit cell is compatible with one virus particle. Rotation function calculations revealed the orientations of the icosahedral symmetry axes. A polyalanine model of the homologous Turnip yellow mosaic virus was used for structure determination by molecular replacement using the program AMoRe. An electron density map computed with 661,311 independent reflections clearly showed density for most of the side chains. X-ray diffraction data for a crystal of the top component were collected at room temperature from a single crystal. The crystals were found to belong to space group R3 with cell parameters a=325.76 Å, b=325.76Å, c=738.94Å α= β =90°, γ=120°. The data consisting of 87.5% of theoretically possible reflections to 3.9 Å had an R-merge of 14.0%. An initial model of the structure was obtained by AMoRe. The top and bottom component structures have been refined to R-factor of 24% and 30% respectively. The refined models will provide the molecular basis required for the rational understanding of the architecture, stability, and assembly of PhMV and plausible mode of RNA encapsidation.
