JOURNAL OF VIROLOGY, Oct. 2008, p. 9546–9554
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 19
Structure of Flexible Filamentous Plant Viruses?
Amy Kendall,1Michele McDonald,1Wen Bian,1Timothy Bowles,1Sarah C. Baumgarten,1Jian Shi,2†
Phoebe L. Stewart,2Esther Bullitt,3David Gore,4Thomas C. Irving,4Wendy M. Havens,5
Said A. Ghabrial,5Joseph S. Wall,6and Gerald Stubbs1*
Department of Biological Sciences and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 372351;
Department of Molecular Physiology and Biophysics and Center for Structural Biology, Vanderbilt University, Nashville,
Tennessee 372322; Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 021183;
BioCAT, CSRRI, and BCPS, Illinois Institute of Technology, Chicago, Illinois 604394; Plant Pathology Department,
University of Kentucky, Lexington, Kentucky 405465; and Biology Department,
Brookhaven National Laboratory, Upton, New York 119736
Received 29 April 2008/Accepted 17 July 2008
Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with
those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron
microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean
mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution
structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous
plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per
Flexible filamentous plant viruses include at least 19 recog-
nized genera (22), almost all in three families of single-
stranded, positive-sense RNA viruses, the Potyviridae, the
Flexiviridae, and the Closteroviridae. Members of the family
Potyviridae account for almost a third of the total known plant
virus species (22) and are responsible for more than half the
viral crop damage in the world (37), infecting most economi-
cally important crops (32). Members of the family Flexiviridae
(2), and particularly of the large genus Potexvirus, are also of
considerable significance to agriculture (42). Both families
show great potential for biotechnological applications, includ-
ing protein expression and vaccine production (12, 54). De-
spite their importance, however, little is known about the struc-
tures of any of the flexible filamentous plant viruses, in sharp
contrast to the amount of data on the rigid tobamoviruses (48,
63) or the icosahedral plant viruses (15); flexibility, instability,
and in many cases low levels of expression have made these
viruses particularly intractable to structural studies. Structural
and evolutionary relationships among the flexible filamentous
plant viruses have been suggested (18, 47, 56, 60), but there is
very little sequence homology between the coat proteins of
viruses in the different families, and there has hitherto been no
structural support for such relationships at the level of either
viral symmetry or coat protein folding. Indeed, reports of viral
symmetry until now appeared to contradict hypotheses of evo-
Soybean mosaic virus (SMV) is a potyvirus, that is, a mem-
ber of the genus Potyvirus, the largest genus in the family
Potyviridae (3). SMV is a major pathogen of soybeans, trans-
mitted efficiently through seed and by aphids in a nonpersistent
manner; yield losses as high as 35% have been reported (30).
Despite dramatic morphological differences, members of the
family resemble the icosahedral plant comoviruses and animal
picornaviruses in genomic organization and replication strat-
egy (32). Early electron microscopic observations found the
potyviruses to be about 7,500 Å long and 120 Å in diameter,
with helical pitches of about 34 Å (44, 60). A fiber diffraction
study (51) of the tritimovirus wheat streak mosaic virus
(WSMV) suggested that WSMV has 6.9 subunits per turn of
the viral helix, but there was considerable ambiguity about that
symmetry determination, which was made from a very poorly
oriented sample. Almost nothing is known about the three-
dimensional structures of members of the family at the atomic
or even at the subunit level.
Potato virus X (PVX) is the type member of the genus
Potexvirus. Virions of potexviruses are flexous rods, 4,700 to
5,800 Å long and about 130 Å in diameter (58). As early as the
1930s, Bernal and Fankuchen (8) used fiber diffraction meth-
ods to determine the helical pitch of PVX to be approximately
33 Å. All potexviruses are believed to share a common archi-
tecture, with slightly less than 9 protein subunits per helical
turn (55). More-precise fiber diffraction studies (52) found the
helical symmetry of PVX to be 8.9 protein subunits per turn
and the helical pitch to be 34.5 Å. The virion was shown to
have a deeply grooved surface. Fourier transform infrared
spectroscopy studies of PVX suggest that the virion surface is
highly hydrated and that the bound water molecules help to
maintain the surface structure of the virion (6). Spectroscopic
studies show that coat proteins of potexviruses and potyviruses
have similarly high ?-helical contents (5, 31).
We have obtained well-oriented fiber diffraction data from
SMV. These data have enabled us to determine a radial density
distribution for the virus and, together with data from scanning
* Corresponding author. Mailing address: Department of Biological
Sciences, Vanderbilt University, Box 351634, Station B, Nashville, TN
37235. Phone: (615) 322-2018. Fax: (615) 343-6707. E-mail: gerald
† Present address: Division of Biology, California Institute of Tech-
nology, Pasadena, CA.
?Published ahead of print on 30 July 2008.
transmission electron microscopy (STEM) and cryo-electron
microscopy (cryo-EM), to determine the viral symmetry.
Cryo-EM data, interpreted using constraints from fiber diffrac-
tion, have allowed us to determine the virus structure, provid-
ing the first reconstructed image of a potyvirus showing subunit
shape and arrangement at any resolution. For comparison, we
have determined the structure of PVX at a comparable reso-
lution and confirmed the PVX symmetry. Both the symmetries
and the structures strongly indicate a relationship between the
MATERIALS AND METHODS
Virus purification. SMV strains G6 and G7 (16) were purified as previously
described (11). The ratios of the optical densities at 260 and 280 nm were close
to 1.2, confirming that coat protein and RNA were present in the expected ratios.
Purified virus was stored at 4°C in 50 mM sodium phosphate, pH 7.6. Most
experiments were carried out using the G7 strain, but fiber diffraction experi-
ments used both strains, and because of the better orientation and higher quality
of the diffraction data, data from the G6 strain were used in the analysis. The
coat proteins of the two strains differ at only 3 amino acid residues (33), and the
substitutions are all conservative and within 20 residues of the N terminus,
known to be located at the outer surface of the virion and probably disordered
(5, 56). PVX was purified and stored as previously described (52).
Fiber diffraction. Dried fibers of SMV were prepared by suspending a 5-?l
drop of virus solution (up to 40 mg/ml in concentration) between two glass rods
?1.5 mm apart and allowing it to dry over a period of hours to days. Humidity
control was essential during drying; even brief exposure to low humidity dramat-
ically reduced order in the fibers. Fibers were made in closed chambers (46) in
the presence of either saturated potassium sulfate or saturated sodium tartrate
solution, producing 97% or 92% relative humidity, respectively.
Fiber diffraction data were collected at the BioCAT beam line of the Ad-
vanced Photon Source synchrotron, Argonne National Laboratory. Fibers were
dusted with calcite, and wide-angle specimen-to-detector distances were deter-
mined from the 104 calcite diffraction ring at a 3.0355-Å resolution (19). Low-
angle specimen-to-detector distances were determined by measuring diffraction
patterns from tobacco mosaic virus (TMV) (35). Wide-angle (to about 4-Å
resolution) and low-angle (to a lowest resolution of about 130 Å) data were
collected and scaled together (34).
Diffraction patterns were analyzed using the program WCEN (9) to determine
experimental parameters and the helical repeat, to apply corrections to the
intensities, and to transform the data from detector to reciprocal space.
Equatorial amplitudes were determined by the method of angular deconvo-
lution (41, 49).
Radial density distributions were determined by applying a Fourier-Bessel
transform to the equatorial data (14). This calculation requires that signs be
applied to the diffraction data; these signs are not directly obtainable from the
data. Signs were determined by the minimum-wavelength principle (10), assum-
ing the radius determined by electron microscopy. The radius is not critical; in
most cases, including that of SMV, large errors in the radius will have no effect
on the sign determination. Essentially, the principle states that for a structure of
a given linear dimension (in fiber diffraction, the particle radius), the Fourier
transform (the Fourier-Bessel transform in fiber diffraction) cannot oscillate at a
frequency higher than that calculated. In consequence, diffracted amplitude
maxima that are sufficiently close together must have alternating signs. The
minimum-wavelength principle was used by Caspar (13) in TMV studies, and the
signs determined for TMV were subsequently shown by isomorphous replace-
ment to be correct (26, 48).
Electron microscopy. Cryo-EM grids were prepared by applying ?3 ?l of
samples to 2/2 C-flat grids (ProtoChips Inc.). The excess liquid from the droplet
was manually blotted away with filter paper, and the sample grid was immediately
plunged into liquid ethane cooled by liquid nitrogen, using a homemade vitrifi-
cation device. Cryo-EM images were collected using an FEI Tecnai 12 (120-kV)
electron microscope equipped with a Gatan cryo holder and a Gatan UltraScan
1000 (2Kx2K) charge-coupled-device camera at a nominal magnification of
?67,000 (1.47 Å/pixel). The defocus values of the micrographs were determined
using the program CTFIT from the EMAN program suite (40), and these values
were used to correct for the phase component of the contrast transfer function
by phase flipping (24). A low-pass filter that truncated the data at 7 Å was also
Three-dimensional reconstruction of virion images from cryo-electron micro-
graphs used iterative helical real-space reconstruction (IHRSR) (20, 21). The
virions are well suited to helical reconstruction, being homogeneous and regular
in structure. IHRSR, which is based on single-particle reconstruction methods,
appears to be the most effective method of reconstruction for these viruses; the
flexibility of the viral filaments makes Fourier-Bessel methods, which require
straight particles with a high degree of order over long distances in the filament,
less useful. Segments of virions were selected using the program BOXER, also
from the EMAN suite; these segments were then used in IHRSR reconstructions
using the SPIDER software package (25). Models were constrained to have
minimum radii of 15 Å (based on the radial density distributions from fiber
diffraction) and maximum radii at least 5 Å greater than maxima from the radial
density distributions. The resolution of the final reconstructions was determined
by Fourier shell correlation (29) of randomly generated half sets of the data,
using a threshold value of 0.5. UCSF Chimera (53) was used to generate models
and density maps.
For electron microscopy phase determination, particles were selected and
processed using the Straighten algorithm (36), a part of the ImageJ software
package (1). Diffraction patterns (Fig. 1) were calculated from selected filament
segments, and the phase differences between symmetrically equivalent points in
the first intensity peak on the first layer line were determined. Only patterns
including clear, symmetric first-layer line intensities were used.
STEM. STEM was used to determine the mass per unit length of the SMV
virions. Grids for STEM were prepared by the wet-film technique (61, 62).
Briefly, titanium grids coated with a thick, holey film were placed on a floating
thin carbon film prepared by ultrahigh vacuum evaporation onto freshly cleaved
rock salt, picked up one at a time so that a thin layer of liquid was retained, and
washed extensively. TMV was allowed to adsorb to the carbon film for 1 min.
After further washings, the sample was allowed to adsorb for 1 min. After
additional washes, the grid was blotted to a very thin layer of liquid, plunged into
liquid nitrogen slush, transferred to an ion-pumped freeze drier, freeze dried
overnight, and transferred under vacuum to the microscope. Images were
analyzed using the PCMass software available from the Brookhaven STEM
website (www.biology.bnl.gov/stem/stem.html). Mass-per-unit-length mea-
surements were calibrated against measurements from the TMV internal
Mass spectrometry. Samples were mixed on-target with matrix containing
40 mg/ml ferulic acid in 60% acetonitrile, 0.1% trifluoroacetic acid, and
subjected to matrix-assisted laser desorption/ionization–time-of-flight mass
spectrometry using an Applied Biosystems Voyager DE-STR mass spectrom-
eter. Data were acquired with delayed extraction (150 ns) in positive-ion
mode (accelerating voltage, 25,000 V; grid voltage, 95%), using the linear
geometry. Masses were externally calibrated using a protein standard mixture
consisting of insulin, cytochrome c, apomyoglobin, aldolase, and albumin.
SDS-PAGE. SMV coat protein was separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) using an 18% polyacryl-
amide gel and visualized by Coomassie blue staining. Gel analysis was per-
formed with Bio-Rad Quantity One software, version 4.6.3.
FIG. 1. Example of a calculated diffraction pattern from a segment
of SMV in a cryo-electron micrograph. The arrow indicates the first
intensity peak in the first layer line. The position of this layer line
corresponds to a spacing of about 165 Å; near-meridional layer lines
corresponding to a spacing of 33 Å are also clearly visible. For phases
to be used in symmetry determination, the first-layer-line intensities in
the four quadrants were required to be clearly visible and symmetric in
position and appearance.
VOL. 82, 2008STRUCTURE OF FLEXIBLE FILAMENTOUS PLANT VIRUSES9547
Helical symmetry. Fiber diffraction patterns from the G6
strain of SMV are shown in Fig. 2A and C. The patterns are
well ordered, with disorientations of about 5° and data extend-
ing to resolutions of 4 Å or better. For comparison, a diffrac-
tion pattern from the potexvirus Narcissus mosaic virus
(NMV) (34) is also shown (Fig. 2B). The patterns show that
the fibers are noncrystalline, with continuous diffraction along
layer lines. The noncrystallinity is confirmed by the absence
of crystalline reflections in the low-angle diffraction pattern
The SMV diffraction patterns are characterized by a series
of equally spaced near-meridional layer lines (seven are visible
in Fig. 2A, and one is visible in Fig. 2C), whose spacing cor-
responds to a helical pitch p of 33.0 ? 0.2 Å. Numerous other
layer lines are also present; the first (Fig. 2A) is at a reciprocal
spacing of (0.2 ? 0.02)/p, corresponding to five times the he-
lical pitch. All of the layer lines can be indexed using this
spacing. If the number of subunits per turn of the viral helix (u)
is uiplus ?u, where uiis an integer and ?u is between 0 and 1,
these layer line spacings imply that ?u is 0.2 ? 0.02 or 0.8 ?
0.02. From the positions of the first maxima on the higher-
order layer lines, it is immediately evident that ?u must be 0.8
rather than 0.2; this conclusion is particularly supported by the
strength of the 19th and 24th layer lines near the meridian,
making obvious pairs with the 20th and 25th layer lines (the
crossover point in the characteristic “X” shape of helical dif-
fraction). If ?u were 0.2, these pairs would include the 21st and
26th layer lines rather than the 19th and 24th (14, 34). Since
the crossover is close to the fourth and fifth near-meridional
layer lines, it follows from the selection rule for helical diffrac-
tion and the properties of Bessel functions (14, 17) that the
integer uiis close to 8.
More information about uican be obtained from diffraction
patterns calculated from cryo-EM images (Fig. 1). The order
of the Bessel function contributing to the first layer line in a
diffraction pattern is the integer nearest to the number of
subunits in each turn of the diffracting helix (14, 17). Its parity
can be determined by measuring the phase difference between
equivalent points in the first intensity maximum on opposite
sides of the meridian. In the absence of noise, a phase differ-
ence of 0° implies that the Bessel order is even; 180° implies
that it is odd.
Phase differences between equivalent points in a given dif-
fraction pattern were measured for an average of 14 separate
points within the prominent first intensity peak on the first
layer line in each of five diffraction patterns from separate
images of SMV. For every point, the difference was greater
than 90°; only three points had phase differences less than 120°,
and the mean phase difference for each image was greater than
145°. The overall mean phase difference was 155°. For PVX,
the overall mean phase difference from 10 points in each of 10
diffraction patterns was 151°. The observed differences clearly
indicate that for both viruses, the number of subunits per
helical turn is closest to an odd integer. In the case of PVX, this
confirms previous determinations of potexvirus symmetry as a
little less than 9 subunits per turn (52, 55), although it is not
consistent with the most recent determination (34) of the sym-
metry of NMV. The direct measurements from cryo-EM are
unambiguous, whereas symmetry determination from fiber dif-
fraction data alone can be very difficult, so it is most likely that
the symmetry of NMV is 8.8 subunits per turn, close to the
PVX symmetry of 8.9 subunits per turn.
An independent determination of helical symmetry is avail-
able from STEM measurements. Fiber diffraction and STEM
are complementary experimental approaches; fiber diffraction
leaves some ambiguity in the value of uibut can determine ?u
very precisely and accurately. STEM, in contrast, is not as
FIG. 2. (A) and (B) Wide-angle fiber diffraction patterns from the potyvirus SMV (A) and the potexvirus NMV (B), with intensities corrected
and data transformed into reciprocal space. The arrow in panel A indicates the first layer line. (C) Low-angle data from SMV, corrected and
9548 KENDALL ET AL.J. VIROL.
precise but provides an unambiguous estimate of u. STEM
calculations require an accurate estimate of molecular mass.
From mass spectrometry, the dominant protein subunit mass
was found to be 29,749 ? 43 Da. This mass is slightly smaller
than the intact subunit molecular mass of 29,859 Da (33),
suggesting that approximately one amino acid had been pro-
teolytically removed. SDS-PAGE analysis of SMV (Fig. 3)
showed that most of the protein was in a single band at ap-
proximately the mass of the intact protein. Minor bands were
seen at about 92% and 88% of the major-band mass; these
bands were estimated to contain 10.6% and 2.1% of the visu-
alized protein, respectively. From these data, we estimate that
the average subunit molecular weight is 98.9% of the major-
band molecular mass. This percentage should be taken as a
maximum value, since it does not consider smaller degradation
products whose bands are too weak to observe, and it does not
consider the possibility that the strongest SDS-PAGE band
includes not only the dominant molecular species but also
slightly smaller degradation products. We assume from the
RNA sequence (GenBank accession no. AY216010) and the
virion length that each protein subunit binds five nucleotides.
For STEM analysis, the mass of one subunit was therefore
taken to be 31,027 Da (98.9% of 29,749), with five nucleotides
with an average molecular mass of 321 Da.
Mass per unit length was determined from STEM measure-
ments of 735 segments of SMV particles to be 7880 ? 400
Da/Å. From this value, the subunit mass, and the helical pitch
of 33.0 Å, u was determined to be 8.38 ? 0.5 subunits per turn
of the viral helix. The helical pitch was taken from the fiber
diffraction measurement because uncertainties in the calibra-
tion of the fiber diffraction measurements are much lower than
those of the cryo-EM measurements, although it is possible
that the pitch changed slightly under cryo-EM conditions. The
value of 8.38 is probably a low estimate of u because of the
effect of protein degradation on the average subunit molec-
ular mass, so the STEM results strongly support the value of
8.8 for u.
This symmetry differs from the reported symmetry of an-
other member of the family Potyviridae, WSMV (51), but the
WSMV symmetry was determined using an almost completely
disoriented fiber diffraction sample and was acknowledged at
the time to be uncertain. The symmetry is in surprisingly good
agreement with early estimates of 7 to 9, based solely on
sedimentation coefficients of the coat protein (27, 45). By anal-
ogy with the observations reported here, we may conclude that
all members of the family Potyviridae have slightly less than 9
subunits per turn of the viral helix.
Virus structures. A radial density distribution for SMV at a
resolution of 22 Å is shown in Fig. 4. This distribution was
determined by the minimum-wavelength principle (10), using
data from the low-angle diffraction pattern (Fig. 2C) at reso-
lutions between 110 Å and 65 Å and the wide-angle pattern
(Fig. 2A) at resolutions between 65 Å and 22 Å. The origin
peak (data inside the equatorial node at 110 Å) was calculated
from a solid cylinder, scaled in amplitude to the low-angle
amplitudes and radially to the first node position (26, 34). The
positions of the peaks in the distribution are probably influ-
enced by series termination error; they remain even when very
large temperature factors are imposed upon the equatorial
amplitudes, but their positions depend on the resolution of the
input data. The maximum and minimum radii, however, ap-
pear to be well determined.
For IHRSR, 18,458 virion segments were selected from the
SMV micrographs and 9,982 segments from PVX. Fewer seg-
ments of PVX were needed for analyses at a comparable res-
olution, perhaps because the PVX virions are better ordered
than those of the more flexible SMV. For SMV reconstruc-
tions, data from 80 micrographs at defocus values between 0.31
and 1.31 ?m were used. For PVX, 132 micrographs were used,
with defocus values between 1.09 and 1.74 ?m. Initial models
were usually featureless cylinders (21) but also included com-
FIG. 3. SDS-PAGE from SMV. Two bands from degradation prod-
ucts of SMV coat protein are clearly visible.
FIG. 4. Radial density distribution in SMV. Units of electron den-
sity (?) are arbitrary.
VOL. 82, 2008 STRUCTURE OF FLEXIBLE FILAMENTOUS PLANT VIRUSES9549
pletely unrelated structures (64). Reconstructions were started
from many different initial symmetries and allowed to continue
until convergence was achieved. Regardless of the initial
model, the final refined symmetries converged to the same
point (Fig. 5) and the refined models were indistinguishable in
SMV reconstructions that were started with symmetries be-
tween 8.7 and 9.0 subunits per turn all converged to a symme-
try of 8.77 subunits per turn (Fig. 5). This value differs from the
fiber diffraction value by slightly more than the experimental
error and may reflect a real difference in symmetry under
different conditions. The difference is not, however, sufficiently
large to affect conclusions about the virus structure. SMV
reconstructions started with symmetries close to 7.8 or 9.8
subunits per turn were not stable, providing further support for
the conclusion that u is close to an odd number. Reconstruc-
tions started close to the correct symmetry are expected to
converge to that symmetry (21). PVX reconstructions started
with symmetries between 8.84 and 9.0 subunits per turn
converged to 8.89 subunits per turn, consistent with previous
determinations (52, 55). Fourier shell correlation plots (29)
(Fig. 6) suggest that the resolutions of both the SMV and
PVX reconstructions are about 14 Å. The refined models
are shown in Fig. 7, together with typical particle images of
SMV and PVX.
The radial density distribution of SMV (Fig. 4) suggests that
the virion has a central hole of radius about 15 Å, slightly
smaller than the central holes of tobamoviruses (48) and po-
texviruses (34). The minimum radius is not clear from the
cryo-EM reconstructions; the density maps are noisy at a very
FIG. 5. Convergence of the rotation angle ? as a function of cycle number during IHRSR. Refinements were started from many different
rotation angles; provided that the angle was not too far from the final refined angle (21), they all converged to the same value. Most of the
refinements used solid cylinders as initial reference volumes, but for SMV, arbitrary unrelated density distributions (solid inverted triangles, open
diamonds, and solid diamonds) were also used.
FIG. 6. Fourier shell correlation plots for the refined PVX and SMV models. In both cases, the correlations fall below 0.5 at a resolution of
about 1/14 Å.
9550KENDALL ET AL.J. VIROL.
FIG. 7. (A) Cryo-electron micrograph of SMV with contrast reversed. Scale bar, 250 Å. (B) IHRSR reconstruction of SMV, section normal to
viral axis. Scale bar (also applies to panels C and D), 50 Å. (C) IHRSR reconstruction of SMV, outside surface view. (D) IHRSR reconstruction
of SMV, section through viral axis. (E) Cryo-electron micrograph of PVX with contrast reversed. Scale bar, 250 Å. (F) IHRSR reconstruction of
PVX, section normal to viral axis. Scale bar (also applies to panels G and H), 50 Å. (G) IHRSR reconstruction of PVX, outside surface view.
(H) IHRSR reconstruction of PVX, section through viral axis. Color coding in panels B, C, D, F, G, and H is from red-orange (low density) to
green-blue (high density).
VOL. 82, 2008 STRUCTURE OF FLEXIBLE FILAMENTOUS PLANT VIRUSES9551
low radius. The maximum radius from both the radial density
distribution and the cryo-EM reconstruction (Fig. 7B and C) is
about 70 Å, greater than most, although not all, previously
reported potyviral radii (37). Previous reports have been based
on negative-stain electron microscopy, and it is likely that neg-
ative stain would penetrate the outer surface of the virus,
leading to underestimates of the radius.
The maximum radius of PVX from the cryo-EM reconstruc-
tion (Fig. 7F and G) is about 65 Å, consistent with earlier
The protein subunits in both the SMV and the PVX recon-
structions from cryo-EM (Fig. 7B, D, F, and H) are compact
and well defined, with a major domain at high radius in the
virion and smaller regions of density reaching into the viral
center. The subunits have their longest dimensions running
approximately radially, reminiscent of the TMV protein sub-
units (48) but much shorter and wider. The SMV subunits are
about 55 Å long, 35 Å wide at the widest (high-radius contact)
point, and 33 Å in the axial direction. The PVX subunits have
similar azimuthal and axial dimensions but are only about 45 Å
long. In both viruses, axial intersubunit contacts are almost
entirely confined to the high-radius region of the structure.
Azimuthal contacts extend more to low radii but are still pre-
dominantly at high radii. The viral structures are very open at
low radius (Fig. 7D and H), consistent with flexibility.
The viral RNA is not expected to be distinguishable from the
adjacent protein at this resolution. If there are five nucleotides
bound to each protein subunit, and if the RNA chain is ex-
tended to about the degree found in TMV, we would expect it
to be located at a radius of about 30 to 35 Å in both viruses,
close to the middle of the protein subunit and at a slightly
lower radius than the major high-radius density. The density in
this region (Fig. 7B and F) is consistent with such a location for
the RNA, but it would be inappropriate to draw any firm
conclusions about RNA structure at the resolution of these
Structure determination of filamentous viruses. In recent
decades, there has been great progress in determining icosa-
hedral virus structures. Studies of filamentous viruses, how-
ever, with the exception of the rigid tobamoviruses (63) and the
filamentous bacteriophages (43), have made little progress. A
hybrid methodological approach to structure determination
such as we have described here appears to offer at least a
partial solution to this problem. The combination of cryo-EM
and X-ray fiber diffraction, supplemented by information from
other sources, has allowed us to determine viral symmetries
and begin the process of three-dimensional structure determi-
nation. Reconstruction from cryo-EM data has the potential to
provide even higher-resolution models, which will be invalu-
able for phasing and eventually exploiting the full potential of
the fiber diffraction data.
Structures of SMV and PVX. We have determined the he-
lical symmetry of SMV to be 8.8 subunits per turn; by impli-
cation, potyviruses in general are expected to have a little less
than 9 subunits per helical turn. We have also confirmed that
PVX has 8.9 subunits per turn. SMV and PVX have diameters
of 140 Å and 130 Å, respectively, and share an open architec-
ture, consistent with viral flexibility. The viruses have very
similar density distributions at low resolution, but there are a
number of small but significant differences.
The most distinct structural differences between the two
viruses are near the outer surfaces of the virions, where the
grooves in the SMV surface are shallower, the virion diameter
is greater, and there is a distinct curvature in the shape of the
subunit by comparison with the PVX subunit. The outer sur-
face is the location of the N terminus of the coat protein (5, 7,
56), the region known to be involved in vector transmission of
potyviruses (4, 37). The structural differences are therefore
consistent with functional differences in this part of the virion.
Several groups have attempted to predict three-dimensional
structures for both potex- and potyviral coat proteins (5, 50,
56). The predicted structures are variations and extensions of
the four-helix bundle structure of TMV (48), consistent with
the high ?-helical content of the proteins and the known lo-
cation of the N and C termini (5, 7, 56). The most detailed
models (5, 50) consist of two domains, an inner four-helix
bundle and an outer ?/? domain, with extra ? strands in the
larger potyviruses. Although these models are similar in shape
to the subunits that we observe, their lengths appear to be at
least 70 Å, significantly too large to fit the observed dimensions
of the virions or the subunits.
Structural relationships among filamentous plant viruses.
The SMV diffraction pattern is very similar to the PVX dif-
fraction pattern (52) and remarkably similar to that of NMV
(34), with similar intensity distributions extending to the limits
of the patterns at a resolution of about 4 Å (Fig. 2). The
striking similarities between potexvirus and potyvirus symme-
try, low-resolution structural models, and higher-resolution fi-
ber diffraction data provide direct evidence for a structural
relationship between the coat proteins of members of the fam-
ilies Flexiviridae and Potyviridae.
It is reasonable to speculate that most or all flexible filamen-
tous plant viruses are structurally related. We note that the
helical pitches reported for the closteroviruses (59) and con-
firmed by our own unpublished results are similar to those of
the potexviruses and potyviruses. A similar speculation can be
applied to rigid, rod-shaped plant viruses, although there is
little or no evidence that the flexible viruses and the rigid
viruses have the same coat protein fold (18, 60). One apparent
exception to these predictions might be the very different re-
ported helical symmetries of the rigid tobamoviruses and
tobraviruses (23, 28). However, reports of the tobravirus symme-
try (about 25 subunits per turn) all appear to depend on a
single early electron microscopic observation, whereas stoichi-
ometric calculations similar to those that have been used for
the potexviruses (38) and potyviruses (51) suggest that the
tobraviruses have only about 16 subunits per turn, similar to
the tobamoviruses. The existence of a common protein fold
will greatly facilitate the design of modified coat proteins for
use in peptide expression (12, 54) and conferral of resistance
on hosts (39, 57) and has important implications for the tax-
onomy, evolution, and further structural study of filamentous
We thank the staff of BioCAT for help with fiber diffraction data
collection, David Friedman and Dawn Overstreet of the Vanderbilt
9552 KENDALL ET AL.J. VIROL.
University Mass Spectrometry Research Center for the mass spec-
trometry data, Martha Simon for help with the STEM data acquisition,
Ian McCullough for help with PVX purification, Hayden Box for help
with molecular graphics, and Ed Egelman for help with IHRSR.
IHRSR computations were carried out using the resources of the
Advanced Computing Center for Research and Education at Vander-
This work was supported by NSF grant MCB-0235653 to G.S. and
USDA-NRI grant 2006-01854 to S.A.G. Fiber diffraction data analysis
software was from FiberNet (www.fiberdiffraction.org), supported by
NSF grant MCB-0234001. Use of the Advanced Photon Source was
supported by the U.S. Department of Energy under contract W-31-
109-ENG-38. BioCAT is an NIH-supported Research Center (RR-
The content is solely the responsibility of the authors and does not
necessarily reflect the official views of the National Center for Re-
search Resources or the NIH.
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