X-ray differaction studies on belladonna mottle virus at 3.8 A resolution

Abstract

Tymoviruses are a group of highly infectious plant viruses found in several geographical locations. The type member of this group of viruses, turnip yellow mosaic virus, has been studied in detail regarding its architecture, stability, assembly and disassembly. The early studies on this virus were instrumental in the elucidation of physico-chemical principles underlying the biological assembly of highly symmetrical macromolecules. Although extensive solution low-angle scattering and single-crystal X-ray diffraction studies have been reported on turnip yellow mosaic virus, it is surprising that the three-dimensional structure of none of the tymoviruses has been determined to date. Determination of the three-dimensional structure of a tymovirus will provide the molecular basis required for the rational understanding of the large number of biophysical data available on tymoviruses. Towards this goal, structural studies on a member of the tymovirus group, belladonna mottle virus (BDMV), were undertaken.

The thesis begins with a broad review of the structure, assembly and evolution of isometric viruses. Significant results of X-ray diffraction studies on spherical viruses are highlighted. The literature on tymoviruses and BDMV relevant to this thesis is reviewed (Chapter I).

The objectives of the investigations reported in this thesis were:

a) Purification and crystallization of BDMV, b) Three-dimensional X-ray diffraction data collection on crystals of BDMV to the highest possible resolution, c) Determination of particle orientation using the high-resolution data, and d) Structure determination by different approaches.

The second chapter of the thesis describes the purification and crystallization of BDMV by methods standardized earlier. For most of the work presented in this thesis, BDMV was propagated on Nicotiana glutinosa and purified from infected leaves by differential centrifugation. Empty capsids formed in vivo were separated from the particles containing RNA by sucrose density gradient centrifugation. The purified virus was periodically examined by electron microscopy, absorbance measurements, sedimentation properties and SDS gel electrophoresis.

Crystals diffracting to slightly better than 4.0 Å resolution were obtained by precipitation of the virus (30–40 mg/ml) from 0.1 M sodium citrate buffer, pH 5.5, containing 2.5–3.0% (w/v) polyethylene glycol 8000 and 10 mM dithiothreitol. The crystals belong to the rhombohedral space group R3 with a = 300 Å and ? = 60°. The unit cell contains one molecule of the virus particle.

Chapter III describes the strategies used for recording, scanning and processing of 3.8 Å X-ray diffraction data on BDMV crystals. Due to the pseudo-cubic nature of BDMV crystals, the special precautions that had to be undertaken to avoid indexing errors are described. The crystals were mounted so as to align the rhombohedral [111] direction parallel to the spindle axis. The oscillation range was set to 0.6–0.8°. The crystal-to-film distance was 100 mm.

Of the 95 crystals examined, 48 were found suitable for data collection and a total of 74 A/B pairs of photographs were recorded using these crystals. A total of 44.8° of oscillation was performed with an overlap of 0.1° between neighbouring photographs. The films were digitized on a Joyce-Loebl film scanner at 50 ?m intervals. The digitized images were processed on a PDP 11/44 system using a computer program originally written by M.G. Rossmann, and subsequently modified by I. Rayment and M.R.N. Murthy.

An alternative approach for the determination of crystal orientation suggested by Matthews and coworkers was attempted and was found to be less suitable for virus crystals when compared to Rossmann’s “convolution” procedure.

The results of scaling and post-refinement of the 3.8 Å screenless oscillation data are presented in Chapter IV. Initially, the 74 pairs of A/B films were scaled together. The R-factors between these pairs varied between 6.2–12.8% with a mean value of 9.5%. Subsequently, the reflections on the 74 files obtained after A/B scaling were merged and scaled using a film of intermediate quality as the standard.

Both for A/B scaling and pack-to-pack scaling, a linear scale and an isotropic temperature-like factor were refined. After the determination of scale factors, the cell parameters and crystal orientations were refined by the post-refinement procedure. The missetting angles derived from different photographs recorded from the same crystal were in good agreement.

A total of 265,327 observations were found on the merged file, of which 220,110 were accepted. The data set contained 385 reflections for which only overloaded measurements were available. The merging R-factor for the 150,912 independent reflections was 16.2%, which reduced to 12.0% for the 91,340 reflections with I/?(I) ? 3.0. The final data set with I/?(I) ? 1.5 represents 65% of the theoretically possible data to a resolution of 4.0 Å and 74% to 4.5 Å.

Chapter V deals with the strategies of collecting and processing low-resolution (7.8 Å) X-ray diffraction data on virus crystals using a Nicolet/Siemens area detector system. Data were collected both on native and derivative crystals of BDMV. Area detector frames were processed assuming a triclinic system and subsequently averaged for rhombohedral equivalents. This strategy could be generally useful for crystals with pseudosymmetry.

The area detector native data scaled well with film data and resulted in an R-factor of 12.4% and a correlation coefficient of 0.94.

The symmetry of the virus particle as established by rotation function studies is described in Chapter VI. The anticipated icosahedral symmetry of the virus particle and the particle orientation in the unit cell had already been determined in an earlier study using 6.0 Å film data. However, redetermination of particle orientation with the higher resolution data collected for the present work and use of a locked rotation function provided better estimates of the directions of particle symmetry axes in the crystal unit cell.

Earlier cross-rotation function studies had suggested that the BDMV structure resembles that of southern bean mosaic virus. The results described in this chapter show an even greater degree of similarity between BDMV and cowpea mosaic virus. These results suggested that cowpea mosaic virus is probably a better starting model for the determination of the structure of BDMV by the molecular replacement technique.

The last chapter (VII) gives an account of the various attempts made towards the structure determination of BDMV. Attempts to determine low-resolution phases by ab initio molecular replacement were not successful, probably due to the pronounced non-spherical character of the BDMV capsid. None of the derivatives tested were found suitable for obtaining reasonably reliable phases for initiating phase refinement by molecular replacement.

Some of the problems associated with preparation of the derivatives were traced to the existence of polyamines bound to BDMV RNA and interfering with the cationic heavy atom salts. Hence, attempts were directed towards using cowpea mosaic virus as an initial model for the BDMV structure. The various strategies used for phase refinement and a critical assessment of the progress of molecular replacement are presented.

The electron density map computed after extending the phases to 4.8 Å resolution was found to be uninterpretable. These results were critically examined to understand the reasons for the quality of the map.

Future work on BDMV X-ray structure determination should focus on preparation of heavy atom derivatives using crystals produced after replacing polyamines by inorganic cations, exploring conditions for producing crystals that diffract to near atomic resolution, and combined studies on BDMV and erysimum latent virus, which is also a tymovirus. These studies will reveal similarities and differences between tymoviruses and other spherical viruses and finer details of tymoviral architecture and evolution.