Structural Elucidation of Potexviruses

1. Structural Elucidationof Potexviruses Tim Bowles Advisor: Gerald Stubbs Honors Presentation Spring 2007

2. Potexviruses • Flexuous, filamentous plant viruses that display helical symmetry. • Complexes of RNA and several thousand coat protein (CP) subunits. • 500 nm in length, 13 nm in diameter. • Structural studies are important for • Agriculture • Biotechnology • Model for virology, viral-host interactions Negative-stain electron micrograph of potato virus X (PVX). Scale bar = 100 nm.

3. Fiber diffraction and phasing • Diffracting units are randomly rotated about a common axis: cylindrical averaging. • FD: I=<FF*>=<|F|2> • F has amplitude and phase. • Phase information required for a high resolution structure. • Phase problem is similar to X ray crystallography, but more difficult to determine. • Phase determination: • crystallography of coat protein. • helical reconstruction of virion using cryo-EM. Fiber diffraction of PVX to 5 Å. Parker et al. (2002)

4. X-ray crystallography of PVX-CP • WT crystals do not diffract. • N-terminal flexibility • Proteolysis • Oligomeric heterogeneity • The goals: To clone the CP gene into an expression vector, to construct, express, and crystallize mutants, to conduct synchrotron diffraction experiments, and to determine the structure of PVX-CP.

5. Summary of results • PVX-CP gene cloned into pET14-b. • Five mutants constructed, confirmed by sequencing. PVX-CP HHHHHHSSGLVPR | GSMSAPASTTQPIGSTTSTTTKTAGA Del 1 HHHHHHSSGLVPR | GS APASTTQPIGSTTSTTTATAGA Del 2 HHHHHHSSGLVPR | GS APASTTQPIGS TATAGA Del 3 HHHHHHSSGLVPR | GS AP PIGSTTSTTTATAGA Del 4 HHHHHHSSGLVPR | GS APASTT TATAGA Del 5 HHHHHHSSGLVPR | GS TTSTTTATAGA N-terminal sequence alignment of PVX-CP mutants.

6. Summary of results • Mutants were expressed, purified and crystallized. • Synchrotron diffraction experiments conducted on ~50 crystals including all five mutants. • No diffraction observed A B (A) Crystals of Del 5 in 20% PEG 3350, 50 mM HEPES, pH 8.0 and 200 mM potassium fluoride. Crystals are right hexagonal prisms, approx. 40X40X60 microns. (B) Crystals of Del 2 in 18% PEG 3350, 50 mM HEPES, pH 8.0 and 200 mM potassium acetate. Crystals are right hexagonal prisms approx. 40X40X120 microns.

7. Discussion • Random orientation of disks might be preventing diffraction. • Over the past decade, 1000’s of PVX-CP crystals have not diffracted. • Another approach to phasing FD data is necessary. • Mutants and methods are still useful. • Identification of important regions, including viral-host interactions. • Assembly, disassembly assays to identify key carboxylates.

8. Helical reconstruction • Helical structures are unique. • No tilting required • Many helical reconstruction techniques exist, but they have shortfalls. • Iterative helical real space reconstruction (IHRSR) overcomes many problems (Egelman, 2000). • Helical parameters: phi and z. • Subunits per turn (u/t) = (360/phi)

9. IHRSR Iterative helical real space reconstruction (IHRSR), Egelman (2007)

10. Helical reconstruction of PMV • Papaya mosaic virus (PMV) • Member of potexvirus genus. • Poorly-oriented FD data, virtually nothing known about structure. • The goals: to generate a low-resolution model of a potexvirus using IHRSR, to determine its symmetry, and to develop IHRSR methods in order to phase FD data of other viruses.

11. Particle selection • 1967 particles were selected from 23 micrographs over a defocus range of -0.71 to -1.78 μm. A B Particle selection cryo-EM micrograph of PMV. representative particles, rotated and aligned.

12. Reconstruction results Cycle number vs. rotation per subunit (phi)

13. B A Which model is correct? • Using a modified version of IHRSR, known as ‘dueling,’ 6.75 u/t was determined to be the better answer. • Particle vs. reference projection assignment • Cycle number vs. rotation per subunit (phi)

14. Model of PMV at 21 Å resolution • Sample of windowed, rescaled, rotated, shifted and assigned particles. • Model of PMV generated by back projection of (A). • Model generated by imposing symmetry on (B) • View of (C) along axis.

15. Symmetry ambiguity • Recent ambiguity about potexvirus symmetry: X.7-X.9 u/t. • 8.7-8.9 u/t from optical diffraction of negative-stain EM of many potexviruses. • 7.7-7.9 u/t from FD of narcissus mosaic virus. • 6.7-6.9 u/t from helical reconstruction of PMV. • No possibility contradicts FD data. • Model provides a plausible explanation for past overestimation of u/t by FD and optical diffraction. • Surface porosity - supported by ROA data.

16. Symmetry determination • Symmetry determination by FD requires r: • r is the distance of a feature from the filament axis that produces diffraction. • At very low resolution, r is usually near protein/water interface - the radius of the virus. • r must be estimated from other sources, such as negative-stain EM. • Surface porosity produces internal diffraction, EM overestimates r. • Diffracting object is no longer at the protein/water interface. • Symmetry is likely 6.75 u/t.

17. Future directions • Pursue helical reconstruction to provide phase information, not crystallography. • Use larger data set. • Apply methods to other flexuous, filamentous viruses. • Reconstruction of a potyvirus, soybean mosaic virus (SMV), shows similar symmetry to PMV. • Relatedness among all flexuous, filamentous viruses? • Use a model to phase well-oriented FD data.

18. Acknowledgments • Dr. Barry Crawford, formerly of the Patton lab. • Dr. Andrzej Krezel and Brendan Borin. • Susan Meyn and the CSB. • Dr. Brandt Eichman and his lab, and Dr. Joel Harp. • Jian Shi (Stewart Lab) • Esther Bullitt (Boston Uni.) • The Stubbs lab: Michele McDonald, Hayden Box, Sarah Baumgarten, Elizabeth Lio, Andrew Wilson, and especially Wen Bian, Amy Kendall, Ian McCullough, and Dr. Gerald Stubbs.

19. Symmetry determination by FD • Deducing symmetry from FD: • intensity is proportional to Jn(2πRr) • Jn = Bessel function of order n • R = distance from meridian • r = distance of diffracting object from filament axis • R is directly measured; r must be estimated from other data. • for a virus with u subunits in t turns: l = tn + um • l = layer line • n = Bessel order • m = any integer • Protein/water interface produces very low resolution diffraction, unless the surface is porous, causing internal diffraction.

20. 1: uninduced 2: induced 3: MWS 4: flow-through 5: wash 1 6: wash 2 7: elution 8: wash 3 1 2 3 4 5 6 7 8 SDS-PAGE gel showing expression of PVX-CP Del 2 (red arrow).

23. 1: MWS 2: post Ni-NTA 3: post IEX, thrombin cleavage 4: post GF B 1 2 3 4 Silver-stained SDS-PAGE gel showing purification of Del 3. Red arrow = Del 3. Blue arrow = Del 3 dimer. 200-400 nm scan of PVX-CP Del 3 after purification.

25. The last asymmetric volume generated by back projection was searched for helical parameters. The graphs plot mean-square deviations in density as a function of changes in and z, and find clear minima at = 53.4˚ and z = 5.1 Ǻ.

26. The FSC curve is shown with the 0.5 CC cutoff marked. It corresponds to a spacial frequency of 0.0728 Å-1 and accordingly a resolution of approximately 21 Å.