The structure determination of bacteriophage phiX174 at 3.4 angstrom resolution

Di Xia, Purdue University

Abstract

The bacteriophage $\phi$X174 was purified using sucrose gradient centrifugation. The virus particles banded into two species which have sedimentation coefficients of 114S and 70S, respectively. Both species crystallized in the monoclinic space group P2$\sb1$ with cell dimensions of a = 305.6 A, b = 360.8 A, c = 299.5 A and $\beta$ = 92.89$\sp\circ$. The crystals diffract very well to 2.7 A resolution. The crystal structure was solved by the Molecular Replacement method using Cowpea Mosaic Virus (CpMV) as an initial phasing model. The particle position in the unit cell was initially obtained using the CpMV as a search model and was later refined using modified electron density derived from CpMV model by averaging and phase extension. A set of Babinet opposite phase solutions were confirmed by Isomorphous Replacement method. Phase improvement was achieved by using molecular envelopes derived from an averaged $\phi$X174 electron density map. A comparison is given between spherical and molecular envelopes used in the structure determination. At 3.5 A resolution, the course of the polypeptide F and G can be completely traced in the electron density map while only the C-terminal portion of the J protein was identified. There is no indication of the existence of electron density for the H protein. An atomic model was built into a 3.4 A resolution electron density map. The F and the G proteins turned out to have an eight-stranded anti-parallel $\beta$-barrel fold similar to those observed in many other viral capsid proteins. A difference map between 114S and 70S data showed some densities which were modeled as DNA structure. In an attempt to investigate the structure of the H protein, missing in the electron density map of the whole particle, the H protein and F capsid were purified by disassembling intact virions with 5M urea. Although no crystals were obtained for the H protein, small crystalline materials of the F capsid were obtained. An investigation of the Molecular Replacement averaging at low resolution is presented in conjunction with the use of $\phi$X174 EM model. It is shown that the EM image can be used as an initial phasing model for Molecular Replacement real space averaging. It is also discovered that the rate of phase convergence is faster for strong reflections than for weak reflections, and it is therefore proposed that initial stages of phase refinement should be performed with reflections of large structure factor amplitudes.

Degree

Ph.D.

Advisors

Rossmann, Purdue University.

Subject Area

Biophysics|Biology|Microbiology

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