Structural Elucidationof Potexviruses

Tim BowlesAdvisor: Gerald Stubbs

Honors PresentationSpring 2007

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 interactionsNegative-stain electron micrographof potato virus X (PVX). Scale bar = 100 nm.

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)

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.

Summary of results

PVX-CP HHHHHHSSGLVPR | GSMSAPASTTQPIGSTTSTTTKTAGADel 1 HHHHHHSSGLVPR | GS APASTTQPIGSTTSTTTATAGADel 2 HHHHHHSSGLVPR | GS APASTTQPIGS TATAGADel 3 HHHHHHSSGLVPR | GS AP PIGSTTSTTTATAGADel 4 HHHHHHSSGLVPR | GS APASTT TATAGADel 5 HHHHHHSSGLVPR | GS TTSTTTATAGA

N-terminal sequence alignment of PVX-CP mutants.

• PVX-CP gene cloned into pET14-b.• Five mutants constructed, confirmed

by sequencing.

Summary of results

(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.

• Mutants were expressed, purified and crystallized.• Synchrotron diffraction experiments conducted on

~50 crystals including all five mutants.– No diffraction observed

A B

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.

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)

IHRSR

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

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.

Particle selection

• 1967 particles were selected from 23 micrographs over a defocus range of -0.71 to -1.78 μm.

Particleselection

(A) cryo-EM micrograph of PMV.

(B) representative particles, rotated and aligned.

A B

Reconstruction results

Cycle number vs. rotation per subunit (phi)

Which model is correct?

• Using a modified version of IHRSR, known as ‘dueling,’ 6.75 u/t was determined to be the better answer.A B

(A) Particle vs. reference projection assignment(B) Cycle number vs. rotation per subunit (phi)

Model of PMV at 21 Å resolution

(A) Sample of windowed, rescaled, rotated, shifted and assigned particles.

(B) Model of PMV generated by back projection of (A).

(C) Model generated by imposing symmetry on (B)

(D) View of (C) along axis.

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.

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.

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.

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.

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.

1: uninduced2: induced3: MWS4: flow-through5: wash 16: wash 27: elution8: wash 3

SDS-PAGE gel showing expression of PVX-CP Del 2 (red arrow).

1 2 3 4 5 6 7 8

1 2 3 4

1: MWS2: post Ni-NTA3: post IEX, thrombin cleavage4: post GF

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.

B

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.

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.