J. Mol. Biol. (1967) 27, 17-24

Structure of Broad Bean Mottle Virus

lI.t X-Ray Diffraction Studies

.J.T.FINcR,R. LEBERMAN AND J.E. BERGER~

Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England

(Received 30 January 1967)

Broad bean mottle virus has been crystallized from sodium citrate solutions and the resulting single crystals have been investigated by X-ray diffraction. The crystals are monoclinic, of space group P2,, and with unit cell dimensions a = 530 A, b = 514 A, c = 275 A and p = 114’. The X-ray diagrams show that the virus particles possess icosahedral symmetry and that they are present in the crystal in two orientations which differ by approximately 4’ about an axis close to [loo]. The approximate arrangement of virus particles within the crystal has been determined from the intensity distribution in the X-ray diagrams. The arrangement is a slightly perturbed version of hexagonal close- packing, the virus particles being S-co-ordinated and the interparticle distances ranging from 275 to 290 A.

Low-angle X-ray diffraction photographs have been obtained from solutions of the virus and a comparison made with various spherically symmetric models. Best correspondence is obtained for a hollow sphere with external radius 130 A and internal radius 55 to 60 A.

1. Introduction Broad bean mottle virus is a small spherical plant virus of particle weight about five million, of which 22% is RNA (Paul, 1961; Yamazaki, Bancroft & Kaesberg, 1961). The protein component of the virus can be chemically dissociated into subunits of molecular weight about 20,000 (Yamazaki & Kaesberg, 1963; Miki & Knight, 1965).

An electron microscope investigation by Finch & Klug (1967) using the method of negative staining has shown that the virus particles have a diameter about 260 A and appear to have a central hole which is penetrated by the negative stain. The protein shell of the virus is made up of 180 subunits arranged on the icosahedral surface lattice T = 3. At the surface of the particle these subunits are grouped into hexamers and pentamers around the 32 lattice points forming prism-shaped morphological units which protrude about 15 A from the body of the particle. Comparison of the electron micrographs with those of turnip yellow mosaic virus and its associated RNA-free protein shell indicated that the RNA of BBMV$ may at least in part be associated with the 32 morphological units of the protein shell.

Parallel with this electron microscope study, BBMV has been investigated by X-ray diffraction from single crystals of the virus and from solutions. We report here the results of this work, which establishes the presence of icosahedral symmetry within

t Paper I of this series is Finch & Klug (1967). $ Present address: Centre for Crystallographic Research, Roswell Park, Buffalo, N.Y ., U.S.A. $ Abbreviation used: BBMV, broad bean mottle virus.

2 17

18 J. T. FINCH, R. LEBERMAN AND J. E. BERGER

the virus particle to at least a resolution of 10 A, and shows that there is a central hole of diameter about 100 to 120 A.

2. Material and Methods BBMV was grown in Vi&a fuba L. var. Broad Windsor which was harvested about

3 to 4 weeks after inoculation. The harvested leaves were dipped in a solution which was 0.1 M in diethyldithiocarbonate, 0.1 M in ascorbic acid and pH 7 and they were then minced in a meat grinder. The minced plant material was squeezed through butter muslin and the sap clarified by centrifuging at 10,000 g for 30 min. To the clarified sap was added 0.1 vol. of a solution that was 0.1 M in diethyldithiocarbonate and 1 M in ascorbic acid. To the resulting solution was added Celite (1 g/l00 ml.) and the solution was filtered. The filtrate was then centrifuged for 3 hr at 100,000 g. The virus pellets were suspended in 0.2 M-EDTA pH 7 and, after a low-speed cleaning centrifugation (10,000 g for 30 min), the solution was centrifuged at 140,000 g for 90 min. The virus pellets were resuspended in 0.2 M-EDTA (pH 7) and subjected to alternate high- and low-speed centrifugation until a clear amber-coloured pellet was obtained; this pellet was suspended in 0.1 M-NaCl and used for crystallization.

No crystals were obtained by our usual method of slow precipitation with ammonium sulphate. However, experiments showed that crystallization was possible with sodium sulphate and sodium citrate. The crystals used in this work were grown by adding to a virus solution of concentration about 1 O/e, a half-volume of a saturated solution of sodium citrate adjusted to pH 6.5 with saturated citric acid. Crystals sufficiently large for X-ray study (up to 0.5 mm diameter) grew at room temperature over several days. The best- shaped crystals were elongated parallelepipeds or triangular prisms. The direction of elongation was found from the X-ray diagrams to be parallel to the short (c) axis of the unit cell.

X-Ray diffraction photographs were obtained from the virus crystals using the equip- ment described by Klug, Longley & Leberman (1966). Low-angle photographs from virus solutions were taken with CuKcc, radiation using an evacuated low-angle camera fitted with a bent-quartz focusing crystal; the specimen to film distance was 20 cm.

3. Results of X-ray diffraction from single crystals (a) Unit cell and space group

X-Ray diffraction photographs show that the crystals are monoclinic with unit cell dimensions a = 530 + 5 d, b (the unique axis) = 514 f 5 d, c = 275 f 3 d, /? = 114”. On the assumption that the virus particles are sufficiently close-packed to occupy half the volume of the crystal, there is room for four virus particles of diameter 260 d in the unit cell. Low-angle precession photographs taken in the directions of the cell sides are shown in Plate I. There are no general absences in the reflections, but those of the types hO0 and Ok0 are only present for h and k even, at least in the low- angle precession photographs obtained. The possible space groups are P2 and P2,. We shall show below that the former is incompatible with the size and orientations of the virus particles, and that the space group is thus P2,; the asymmetric unit of the crystal consists of two virus particles.

(b) Symmetry and orientation of the particles

The presence of icosahedral symmetry in spherical virus particles results in spikes of reflections of high intensity in the X-ray diagrams in directions corresponding to the symmetry axes of the particles (Caspar, 1956; Klug, Finch & Franklin, 1957). The spikes are identified by the characteristic angles they make with each other in the various zero layer planes of the reciprocal lattice. Such spikes can be seen fairly

PLATE I. X-Ray precession photographs from single crystals of BBMV token in the directions of the unit cell sides. The equivalent specimen to film distance is 13.5 cm. The directions of the 2., 3- and &fold axes of the icosahedral point group 532 which lie iu or close to the zero layer of the reciprocal lattice in each case are indicated by lines. The intensities of the reflections tend to be strongest in these directions.

(a) [ 1001 axis. Two sets of axes are marked by full and broken lines, respectively. Both sets arise from views of the icosahedral point group in the direction of a 2-fold axis but they differ in orientation by about 4’. The doubling of the spikes of strong reflections is most clearly visible in the directions of the B-fold axes on the left-hand side of the photograph.

(b) [OIO] axis. The set of axes marked corresponds to a view of the point group in the direction of a 2-fold axis.

(c) [OOl] axis. The marked axes correspond to a view of the point group in the direction of a 3-fold axis.

In (b) and (c) the difference between the two orientations of the virus particles within the crystal is not resolved and thus only one set of axes is marked.

[facingp. 18

PLATE Il. X-Ray still photograph from a crystal of BBM,IV taken in a direct,ion close to the zone axis [210]. The equivalent specimen to film distance is 9 cm.

The positions of the zone axes [ 1101, [210] and [ IOO] are marked respectively 9, B and C. The pattern is dominated, however, by the broad circles of reflections about the marked point X. which does not correspond to the position of a rational zone axis, but to that of a vector joining virus particles which are in the same orientation but which are not at equivalent points in the crystal lat,tice.

STRUCTURE OF BROAD BEAN MOTTLE VIRUS 19

clearly in the BBMV X-ray diagrams in Plate I and show no signs of decreasing in intensity at spacings of 10 A, indicating that, at least to this resolution, the virus particle possesses icosahedral symmetry. In the [OlO] precession photograph (Plate I(b)) spikes corresponding to the projection of the icosahedral point group in the direction of a 2-fold axis can be seen; in this projection there is a 2-fold axis the corresponding spike of which in the photograph lies about 2” from the [lOO] direction, a 5-fold axis the spike of which is very close to the [loll direction and a 3-fold axis the spike of which is very close to the [OOl] direction. In the [lOOI precession photo- graph (Plate I(a)), two sets of spikes can be seen, each set corresponding to the 2- fold projection of the point group, the sets differing in orientation by about 4”, sym- metrically disposed about the [OlO] direction; the directions corresponding to the 2- fold axes of the virus particles are thus -& 2” from the [OlO] direction in this pro- jection. The [OOl] precession photograph (Plate I(c)) shows six spikes at 60” to each other, corresponding to the projection of the virus particle in the direction of a 3-fold axis. The positions of the spikes in the various X-ray diagrams are consistent with the presence of virus particles with icosahedral symmetry present in two slightly differing orientations.

(c) Arrangement of virus particles

Examination of the distribution of intensity of the reflections of the [loo] X-ray diagram (Plate I(a)) reveals a tendency towards a smaller unit cell; the reflections with high intensity are confined to bands perpendicular to the [OlO] axis, spaced about 0.01 1(1-l apart. These bands are sections of rather thick layers of reciprocal lattice points on which reflections with strong intensity occur. The direction perpen- dicular to these layers has the quality of a zone axis, and, as can be seen in Plate II, where it is marked X, it has the characteristic appearance of a zone axis in still photographs of virus crystals, except that the circles of spots arising from the inter- sections of the layers of strong reflections with the sphere of reflection are rather broad, corresponding to the thickness of the layers. Indices [O-5, 0.195, O] can be assigned to this pseudo-zone axis from its orientation relative to the crystal axes and the distances between the layers of strong reflections perpendicular to it. These indices define a vector [0*5a + 0*195b] between points in the unit cell where the structure repeats itself approximately, T.e. between two virus particles in the same or nearly the same orientation. The fact that the system of circles corresponding to this pseudo- zone axis on still photographs can be seen clearly to spacings of at least 3 A implies that the virus particles related by this vector must be exactly in the same orientation or at least to within 1” of the same orientation. A combination of this vector with the lattice vectors a and c results in puckered or corrugated sheets of virus particles, parallel to the (GlO) planes, in which all the virus particles have the same orientation as shown in Fig. l(a). In this Figure, the virus particles are represented by icosahedra to indicate approximately the orientations of their symmetry axes (see previous section). Within these sheets, alternate rows of virus particles parallel to the c-axis lie 100 A below the remaining rows (in the direction of the b-axis), and this dis- placement gives rise to the above-mentioned 100 A modulation in the intensities of reflections in the [OlO] direction.

The existence of these corrugated (010) sheets of particles is incompatible with the space group P2 for the following reason. The virus particles within each sheet are in the same orientation but do not have 2-fold axes in the direction of the y-axis;

20 J. T. FINCH, R. LEBERMAN AND J. E. BERGER

they therefore occupy general positions in the unit cell and there is insufficient room for further particles related by a 2-fold crystallographic axis parallel to the y-axis. The space group must therefore be P2,.

(a) (010) (b) (001)

FIG 1. Diagram showing the arrangement of virus particles, represented by icosahedre, in crystals of BBMLV. For clarity, the difference between the two orientations of the virus particles is shown exaggerated approx. 4 times.

(a) Normal to (010) showing a corrugated sheet of virus particles all in the same orientation. (b) Normal to (001) showing how adjacent sheets of the type shown in (a) would be arranged

for closest packing.

Halfway between two corrugated sheets of the type shown in Fig. l(a), which of course are b apart, there must also lie, as a result of the space group, an identical sheet rotated by 180” about the y-axis. We have insufficient information in the present X-ray diagrams to determine the relative translational positions of the particles in these two types of sheet in directions perpendicular to the y-axis, but from packing considerations one would expect the troughs on adjacent corrugated sheets to overlie each other and for particles on one sheet to lie over the mid-point between two particles in the sheet below, as shown diagrammatically in Fig. l(b). On this basis, the four virus particles in the unit cell would occupy the two sets of general positions with co-ordinates close to (0, y, l/4), (0, y + 4, 2) and (4, y + O-195, a), (4, y + O-695, 4) (see International Tables, 1952, p. 79), the value of y being arbitrary.

In this arrangement each virus particle is in contact with the eight nearest neigh- bours, and with six of these it is coplanar (the (100) plane) in an almost regular hexagonal manner. Adjacent (100) planes of virus particles are arranged almost as in hexagonal close-packing, but sufficiently displaced to bring the co-ordination to eight rather than 12.

In the corrugated (010) sheets (Fig. l(a)) all the virus particles make contact in directions close to 3-fold axes; the interparticle distances are 275 A(c) and 283 A. Between these sheets the virus particles make contact almost halfway between S-fold and 3-fold axes, the interparticle distance being close to 290 A.

(d) Intensity distribution in the X-ray diagrams

In the case of turnip yellow mosaic virus (Hlug et al., 1966), it was possible, from a comparison of the intensity distribution in the X-ray diagrams with those calculated for

STRUCTURE OF BROAD BEAN MOTTLE VIRUS 21

various model structures and from the properties of certain classes of reflections, to assign co-ordinates to the centres of mass of the parts of the protein structure units protruding from the main body of the virus particle. Also, by comparing the X-ray diagrams of the bottom (whole virus) and top (empty protein shells) components, it was possible to deduce the gross distribution of RNA within the virus particles and to coniirm this by matching the protein scattering in virus crystals by working in high salt concentration. BBMV is much less suited to such work for, while the crystal structure of turnip yellow mosaic virus results in the intensity of certain classes of reflections being related fairly directly to the scattering function of one virus particle, this is not so for BBMV. Thus the intensity distribution observed for BBMV involves interparticle effects such as the above-mentioned 100 A modulation in the [OlO] direction, and also effects due to the two slightly different orientations of particle. Further, in the absence of a top component of BBMV, the direct effects of the RNA cannot be observed. Bowever, the electron density of saturated sodium citrate is about 0.36 electron/A3-close to that of protein-and thus the low-angle X-ray diagram of a virus crystal in such high salt concentration should be mainly due to RNA.

We have obtained X-ray diagrams from BBMV crystals in,saturated sodium citrate solution and these do show marked differences compared with those shown in Plate I from crystals in one-third saturated sodium citrate. The most prominent effect is the disappearance, at high salt concentration, of the spikes of reflections of high intensity in the directions close to 2-fold axes of the virus particles, although the spikes in the directions corresponding to B-fold axes remain.

In the absence of more data and more precise information on the crystal structure, we have not attempted au interpretation of these changes and of the over-all intensity distribution in terms of the structure of the virus particle. Some information on the spherically averaged radial dimensions of the particle has, however, been more easily obtained from the low-angle X-ray patterns as described in the following section.

4. Low-angle X-ray Scattering from Virus Solutions In order to investigate the radial density distribution within the BBiMV particle,

low-angle X-ray diffraction photographs were taken from solutions of the virus. With a virus solution of high concentration (about 25%), a strong inter-particle pattern was obtained consisting of sharp lines with reciprocal spacings in the ratios 1: 2/3 : 44 : 47 : 1/g, indicating that the virus particles were packed together hexagonally in sheets. In this particular case, the interparticle distance was 290 A; but possible variation of this with concentration was not investigated.

With virus concentrations around 5%, no trace of this inter-particle pattern was observed, and the X-ray diagram obtained corresponds to the spherically averaged intensity of scattering from a virus particle. The intensity distribution obtained is shown in Fig. 2, and this was compared with the corresponding intensity distributions of various spherically symmetric models of uniform density. Strictly, such a com- parison should be limited to the region of the scattering function which is spherically symmetric--the very low-angle region where the resolution is insufilcient to distin- guish angular fluctuations in density at any one radius. For a particle with icosahedral symmetry and of diameter 300 b, the scattering function will only be spherically symmetric at reciprocal spacings less than about 0609 8-l (see Finch & Holmes (1967) and compare the case of turnip yellow mosaic virus (Klug et al., 1966)). However, since the electron micrographs of BBiKV indicate that the outer parts of the protein subunits are fairly uniformly distributed over the surface of the virus particle, we feel justified in tentatively including data beyond 0909 d-l in these considerations.

Since the positions of the first minima in the intensity distribution are likely to be less affected by departures from spherical symmetry than the actual values of the

22 J. T. FINCH, R. LEBERMAN AND J. E. BERGER

intensity, these positions were lirst compared with those of a spherical model of uniform density, namely, a sphere of diameter D in which is a central hole of diameter kD. The positions of the first minima in the scattering function of such a model are quite sensitive to the values of k and D. The variation with k of the positions of the first four minima is shown in Fig. 3, and the best agreement with the minima of the experimental curve is found by the method shown in that Figure for k = 0.4. On the bgsis of this value of k, the positions of the experimental minima correspond to those of a particle of uniform density with spherically averaged external diameter 260 f 10 d and internal diameter about 100 8.

Reciprocal spacing

FIG. 2. Intensity of low-angle X-ray scattering from solutions of BBMV.

In order to relate these dimensions to the virus particle, it remains to be shown that the spherically averaged density of the virus is uniform and does not vary markedly in the radial direction between radii 50 and 130 d. This has been established by calculating the Fourier transform of the amplitudes of scattering in the region 0.005 to 0.02 b-l, and comparing this with the corresponding transforms for various spherically symmetric models of uniform density. The signs of the four intensity peaks in this region must alternate - + - + and the Fourier transform obtained is shown in Fig. 4(a). Since data within the origin scattering peak were not included, the transform obtained is not the spherically averaged radial density distribution, but corresponds to the latter placed on a sloping background which is determined by the origin peak and which, without knowledge of the shape of the origin peak, it would be begging the question to insert. The Fourier transform of the corresponding region of the scattering function of a uniform sphere of diameter 260 8, is shown in Fig. 4(b). In order to minimize fluctuations due to cut-off, the structure amplitudes were in each case multiplied by an artificial temperature factor exp ( -4000R2), which reduced those in the region R = 0.02 B-l by 75% compared with those at 0607 d-l. This had the further advantage of reducing the effect in the transform of the virus data in the region outside that corresponding to spherical symmetry. The difference

I.(

0.i

0.1 -x

0,

0

STRUCTURE OF BROAD BEAN MOTTLE VIRUS

(0)

3

3

5

I

2

3

--R'(=DR)-+

>

23

FIQ. 3. Method of finding the hollow sphere of uniform density which has minima in the scattering function closest to those of BBMV.

(a) The position of the fnst 4 zero values of the scattering curves of hollow spheres with e~tmnal diameter D and internal diameter kD. The abscissae, R’, are the products of D and the reciprocal spa&g, R.

(b) A fan of lines from an arbitrary point P on the k-axis making angles 8 with that axis such that tan 6 is proportional to the reciprocal spacings of the 6rst 4 minima of the scattering curve from a solution of BBMV.

P is moved along the k-axis until, as in the position shown, the intersections with the curves in (a), marked X, are most nearly on a line parallel to the abscissa; in this case the line is k = 04. For other positions of P, for example those indicated by the broken lines, the intersections with the curves marked 0 and A do not lie on such a line.

(b) .,=558,

(4

-.-- q=60ii

I I I I I I I I 1 I 1 I I I I I I 1 I 1 0 100 200 0 100 200

Fro. 4. (a) Fourier transform of the scattering amplitudes of BBMV in the region 0.005 to 0.02 A-1, i.e. with the zero-order peak omitted.

(b) Fourier transform of the corresponding region of the scattering curve of a uniform sphere of radius 130 A.

(c) The difference, (a) - (b), between the above transforms. (d) Similar difference curves between (a) and the corresponding Fourier transforms of hollow

spheres of external radii 130 A and internal radii r, = 40, 60, 55, 60 and ‘70 A.

24 J. T. FTNCR, R. LEBERMAN AND J. E. BERGER

between the two curves scaled in amplitude to minimize the difference in the region 80 to 150 A is shown in Fig. 4(c). This difference curve indicates the nature of the departure of the radial density distribution of the virus from that of a uniform sphere of radius 130 A. The fact that by an appropriate choice of scaling factor the difference curve can be made not to depart markedly from zero at radii greater than 50 8, conf?rms that the spherically averaged radius of the virus particle is close to 130 A. The other main feature of this curve is the central minimum. This, if taken at face value, would indicate the presence of a central hole of radius about 50 A within the virus particle. However, such an interpretation must be treated with caution for two reasons. First, these curves cannot be relied on near zero radius since errors in the calculation accumulate in this region. Second, the central minimum may be affected by the difference between the origin scattering peak of the virus and that of the uniform sphere model. The latter effect will be minimized by replacing the uniform sphere by a model representing more closely the radial density distribution of the virus. The above calculation was thus repeated for hollow spheres with external radii re = 130 B and internal radii r, = 40, 50, 55, 60 and 70 8. The difference curves obtained by subtracting from the experimental curve (Fig. 4(a)) the corresponding curves for the hollow spheres are shown in Fig. 4(d). The curves for r, = 55 and 60 if can be seen to depart very little from zero, showing that the uniform-density models used in these cases adequately represent the spherically averaged virus particle.

We therefore conclude that the spherically averaged external diameter of BBMV is close to 260 A and there is a central hole of diameter 100 to 120 1(1.

One of us (J. E. B.) acknowledges support by an American Cancer Society grant E-445.

REFERENCES

&spar, D. L. D. (1966). Nccture, 177, 475. Finch, J. T. & Holmes, K. C. (1967). Methods in Vi’iroZogy, vol. 2. New York: Academic

Press. Finch, J. T. & Klug, A. (1967). J. Mol. BioZ. 24, 289. Klug, A., Finch, J. T. & Franklin, R. E. (1957). Biochim. biophys. Acta, 25, 242. Klug, A., Longley, W. & Lebermau, R. (1966). J. Mol. BioZ. 15, 315. M&i, T. t Knight, C. A. (1965). Virology, 25, 478. Paul, H. L. (1961). 2. Naturf. 16b, 786. Yamazaki, H., Bancroft, J. & Kaesberg, P. (1961). Proc. Nat. Acd A%., Wash. 47, 979. Yaznazaki, H. t Kaesberg, P. (1963). J. Mol. BioZ. 6, 465.