Structural Elucidation of Potexviruses. Tim Bowles Advisor: Gerald Stubbs Honors Presentation Spring 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.
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Advisor: Gerald Stubbs
Negative-stain electron micrograph
of potato virus X (PVX).
Scale bar = 100 nm.
Fiber diffraction of PVX to 5 Å.
Parker et al. (2002)
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.
(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.
Iterative helical real space reconstruction (IHRSR), Egelman (2007)
cryo-EM micrograph of PMV.
representative particles, rotated and aligned.
Cycle number vs. rotation per subunit (phi)
AWhich model is correct?
5: wash 1
6: wash 2
8: wash 3
1 2 3 4 5 6 7 8
SDS-PAGE gel showing expression of PVX-CP Del 2 (red arrow).
2: post Ni-NTA
3: post IEX, thrombin cleavage
4: post GF
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.
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.