Peter A ~imrnind*, David wild2+ and Jean witz3

Transcription

1 The three-dimensional distribution of RNA and protein in the interior of tomato bushy stunt virus: a neutron low-resolution single-crystal diffraction study Peter A ~imrnind*, David wild2+ and Jean witz3 'Institut Laue-Langevin, B.P. 156, Crenoble Cedex 9, France, 2EMBL, c/o ILL, B.P. 156, Crenoble Cedex 9, France and 31mmunochimie des Peptides et des Virus, UPR 9021 du CNRS, 15 rue Descartes, Strasbourg, France Background: The published high-resolution model of the isometric T=3 plant virus tomato bushy stunt virus (TBSV) shows the packing in three different environments (A, B, C) of the 180 coat protein subunits of the capsid. It does not, however, account for the localization of either the viral RNA or -25% of the amino acids of the protein subunits, although at least the RNA is rigdly linked to the viral capsid. Solution studies have shown that most of the missing protein is located in an inner shell, and that most of the RNA is sandwiched between the two protein shells. Results: We have determined the organization of TBSV at 16 A resolution, using neutron single-crystal diffraction. Connections between the two protein shells are confined to the 20 three-fold axes of the virion, where three C-type subunits meet. Much more RNA density is located under the 30 C-C dimers than under the 60 A-B dimers, where we could even identify lagoons of solvent. Conclusions: Our results emphasize the importance of the amino termini of the 60 C-type protein subunits not only in the RNA-protein interactions but also in the organization of the coat protein, and, probably, in the assembly of the virion. The lack of equivalence between subunits of classes A or B and subunits of class C is even more pronounced in the interior of the virion than in the outer shell, which possesses icosahedral symmetry. Structure 15 December 1994, 2: Key words: contrast variation, neutron crystallography, tomato bushy stunt virus (TBSV), T=3 shell Introduction Tomato bushy stunt virus (TBSV) was the first isometric virus for which the detailed folding of the polypeptide chain of the coat protein was described [I-31, but, as also observed for many other isometric viruses, neither the RNA nor a portion of the protein was visible in the high-resolution maps (reviewed in [4-61). TBSV is made of a T=3 shell built of 180 subunits of the same coat protein (M 40000) and a single-stranded RNA (Mr 1.6 ~ 10~). The 387 amino acids of the coat protein form four different domains: the amino terminus (R; 66 residues), the connecting arm (a; 35 residues), the surface domain (S; 170 residues) and the projecting domain (P; 114 residues) connected to S by a hinge [2]. The core of the S domain possesses the canonical twisted P-sheet fold common to all known coat proteins of simple viruses except that of phage MS2 (reviewed in [4-71). The P domains are grouped into dimers, forming 90 projections at the periphery of the virion. The tightly packed S domains form the surface. The 180 subunits exist in three distinct conformations, A, B and C. Subunit C differs from A and B by the conformation of the hinge separating the domains S and P, and the ordering of the arm. Part of this arm contributes an extra strand to the canonical twisted P-sheet: 30 C-C dimers are centred on the icosahedral two-fold axes, whereas the other 60 dimers are of type A-B and are centred on the quasi-two-fold axes. C-type subunits are connected in groups of three by a p-annulus centered on the three-fold axis of the icosahedron. They form a loose net covering the whole surface of the virion. All R-domains, containing many basic amino acids, as well as the arms of A and B subunits (25% of the protein altogether) appear to be spatially disordered and not visible in the electron-density maps, as is the case with all of the RNA component. NMR [8] has shown, however, that in compact TBSV the RNA is tightly bound to the capsid. Spatial disorder is therefore static and not dynamic, implying that either the internal architecture is not the same in all virions, or a unique architecture has been averaged out in the crystal because contacts between neighbouring particles involve the periphery of the virions (possessing icosahedral symmetry), or both these cases (these arguments have already been developed by Harrison [1,4] and have been described in detail by Bentley et al. [9]). Neutron small-angle scattering studies in solution have shown that in TBSV most of the RNA is sandwiched between two protein shells [lo]. The inner radius of the outer protein shell (1 10 A) coincides well with the average inner radius of the 180 S domains of the capsid. In the 16 A resolution map, obtained from single-crystal X- ray diffraction studies of TBSV, strong density features were also seen below the S domains [I I], but at low resolution the electron density of hydrated RNA does not differ sufficiently from that of protein to allow an unambiguous identification. A similar argument also holds for - - *corresponding author. +Present address: EMBL, Meyerhofstrasse 1, Postfach , D Heidelberg, Germany. O Current Biology Ltd ISSN

2 1192 Structure 1994, Vol 2 No 12 the interpretation of the inner density in three-dimensional models reconstructed from cryo-electron micrographs [12]. Such ambiguities do not exist for scattering length density maps obtained from low-resolution neutron-scattering studies at various contrasts, as the average contrast of protein is very weak in 38-40% D 2 0 buffers, and that of RNA is very weak in 68-70% D 2 0 buffers (for example, see [10]). We undertook this neutron-diffraction study in order to obtain a more precise picture of the three-dimensional organization of the interior of the virions. Indeed, in a preliminary test we found that for a crystal soaked in 40% D 2 0, diffraction spots such as 301 and 310, which are located at the same distance from the origin but are crystallographically unrelated in space group 123, differed in intensity much more than in the X-ray diffraction pattern. This provided evidence, at a resolution as low as -100 A, that the distribution of RNA departs from spherical symmetry. A resolution of 16 A is sufficiently low to permit the use of contrast-variation techniques, and high enough to allow the identification of the regions where, on average, protein and RNA accumulate, although, of course, it does not allow the localization nor the identification of single amino acids or nucleotides. Indeed, it has proved useful in the study of the nucleosome core particle [13]. A similar investigation of the smaller, T=1 virus, satellite tobacco necrosis virus (STNV), has already been published [9]. Our results show that the organization of the interior of TBSV is very different from that of STNV. Furthermore, differences in the packing densities of RNA and protein among the three symmetrically distinct classes of subunits emphasize the importance of C-C type dimers in the RNA-protein interactions and therefore, very probably, in the initial stages of the assembly of the virion. Results General features Neutron scattering length density maps were calculated at four D20/H 20 contrasts (see the Materials and methods section): 0% D 2 0 (100% H 2 0) and 38%, 70% and 100% D 2 0. Fig. 1 shows the same section through the centre of the virus particle for each of these contrasts; in order to be able to place a chemical interpretation on the density in the various maps we also calculated a 'matchpoint map', as described in Materials and methods and shown in Fig. 2. We are thus able to assign to any point in the map a D 2 0/H 2 0 mole fraction at which the contrast would be zero. This match point is then an indication of the chemical species found at that point, i.e. a match point of 70% indicates RNA or clusters of amino acids such as arginine, asparagine and aspartate possessing many exchangeable hydrogen atoms (see the Materials and methods section), whereas 40% indicates protein. Intermediate match points indicate clusters of hydrophilic amino acids whereas lower match points indicate hydrophobic amino acids. Points at which the density is zero or very small at all contrasts will have a very poorly Fig. 1. Section through the centre of the virus particle in 0% D 2 0, 38% D 2 0, 70% D 2 0 and 100% D 2 0. The positions of the icosahedral two-fold (s2), quasi-two-fold (q2), three-fold (s3) and five-fold (s5) axes are indicated in the 70% D 2 0 map. All displayed maps correspond to an average density of zero of the whole unit cell. The density of the solvent corresponds therefore to the minimum density of the 0% D 0 map and to the highest density of the 100% D0 map. The density scale extends from blue (lowest, may be strongly negative) to green, yellow and red (highest).

3 RNA and protein in tomato bushy stunt virus Timmins, Wild and Witz 1193 determined (or indeterminate) match point and correspond to pure solvent. Such regions are indicated in red in Fig. 2. The 0% map of Fig. 1 shows indeed, that there exists density in the interior of the virus particle, although it is usually weaker than that of the outer shell (called the 'coat' hereafter). In this buffer the mean contrast for non-hydrated ordered protein and RNA is high, with that of RNA being -80% higher than that of protein. Preferential hydration of RNA would, however, provide a similar contrast for both moieties. In 70% D20 the mean contrast of RNA is very close to zero whereas that of protein is negative. The 70% map shows that the protein is divided into shells. The outer shell (coat), which formed the initial phasing model from the X-ray structure, is faithfully reproduced. The outer limit of the projection corresponding to the AB dimer possesses local two-fold symmetry about an axis that, in the plane of the map, is inclined by -8.5' with respect to the three-fold axis (see also Fig. 6 of [Ill). Just inside the coat lies a region of very low contrast which must therefore be either RNA or solvent. The outer shell is sharply delimited, and its limits closely follow those of the '70% model map' (see the Materials and methods section) except near the three-fold axis, where the region of low contrast is crossed by a connection with another shell of density extending from A radius with protrusions in places out to -110 A. This connection is even more clearly seen in the match point map (Fig. 2). The inner shell (which has a density approximately half of that of the outer one) also protrudes to a higher radius near the quasi-two-fold axis, but does not connect clearly to the outer shell in this direction. In 38% D20 the mean contrast of protein is very close to zero. Local fluctuations attributable to particular amino acids can, however, produce significant positive or negative density, which may even attain values as hlgh as those for hydrated RNA (see the Materials and methods section). The map in 38% D20 therefore shows features which may be assigned to RNA (high positive contrast) and features characteristic of protein (both positive and negative contrasts). The degree to which features within the protein may be seen, even when on average it is contrast matched, is well illustrated by the density seen in the outer shell. These patches of density, also visible in the '38% model map', indicate areas of particularly hydrophllic (for positive contrast) or hydrophobic (for negative contrast) amino acids (see the Materials and methods section). Most of the density in this map lies in a shell immediately internal to the outer protein shell. Close comparison with the 70% D20 map shows that not all the zero contrast in the 70% map is accounted for by the 38% map, and that remaining areas of zero contrast must therefore correspond to solvent. Such lagoons of solvent occur especially close to the quasi-two-fold axis, i.e. under the ordered part of the A-B dimers (Fig. 2). Fig. 2. Section through the centre of the virus particle, showing the distribution of neutron scattering density match points. The colour code of the match-point scale is indicated at the bottom of the map. Points <55% may be interpreted as protein, 55-70% as RNA and elsewhere as pure solvent (see text for details). The symmetry axes are oriented as for Fig. 1. In 100% D20 all contrasts are negative. RNA possesses a weak contrast whereas that of aliphatic amino acids is the highest. Two more shells, extending from the centre of the virus particle to -30 and from 30 A to 50 may also be seen in the maps. Their interpretation as protein (up to 30 A) and RNA-rich regions is ambiguous as errors in the data accumulate at low radius upon Fourier transformation of ill-defined structure factors at large Q-values. These innermost regions, in any case, account for <5% of the total volume of the virion. The essential features of our model, in particular the presence of lakes of solvent in the intermediate shell containing most of the viral RNA, are illustrated three dimensionally in Fig. 3. Protein structure As the scattering length density of RNA is rather homogeneous at 16 A resolution, the 70% D20 map is a good indication of the protein density. Several interesting features emerge from a close examination of this map. The most striking conclusion is that a very large proportion of the amino acids not found in the high-resolution X-ra study is located in a shell lying between radii of -50 K and 95 A. The density within this shell is not totally homogeneous and tends to confirm the existence of at least partially folded but somewhat disordered R regions.

4 Structure 1994, Vol 2 No 12 Fig. 3. Stereoview of TBSV at 16 A resolution. The contours of the neutron-scattering maps both in 70% D 2 0 (blue) and 38% D 2 0 (red) are shown. An octant has been removed to show the interior of the particle. The distribution of proteins into two shells, as well as the inhomogenous distribution of RNA and lakes of solvent sandwiched between the two protein shells, is clearly visible. Connections between the two protein shells are confined to the three-fold axis (top of octant). The details of the 70% map in the only region where clear connectivity between the outer and inner shells is seen are shown in Fig. 4, together with the trace of the C, chain of the C-subunits that meet at the [3-annulus centred on the three-fold axis. A protrusion extends beyond the ordered part of the 3-annulus and fades out towards smaller radii, whereas three protrusions extend from the inner shell towards the outer one. It suggests that after meeting at the -annulus, the three polypeptide chains separate again before reaching the bulk of the inner shell. (In Fig. 4, maps have been contoured at rather high density levels. In the map drawn at the 2cr contour, the tips of the four protrusions come very close to each other, on the three-fold axis; data not shown.) The connection contains many amino acids because, apart from Lys61, the segment R(57)NRRKQGNQQ(66) is composed of hydrophilic residues that possess only a weak contrast in 70% D 2 0. This connection between the two shells therefore includes residues 57-66, if not 50-66, and contains 5-7 of the 11 basic amino acids of the 66 amino-terminal residues of the C subunit. It is worth noticing that the region contains, apart from Gly63, only residues with basic or amine side chains, a situation reminiscent of that for the first 11 residues of the coat protein of STNV, which also could not be located in the high-resolution electron-density maps [9,14]. This interpretation assumes that the polypeptide chain does not fold back to form this connection, a reasonable hypothesis as none of the first 66 residues could be located in the high-resolution electrondensity maps. The traces of the ordered parts of the polypeptide chains of A and B subunits are lost in highresolution maps near the quasi-two-fold axis, the chains pointing in the directions of the three-fold and five-fold axes respectively. We have carefully checked that the inner delimitation of the 70% D 2 0 map is very sharp and closely follows the trace of the ordered parts of the A-type and B-type protein subunits, without protrusions or extensions to lower radius (data not shown). Many Fig. 4. Stereoview of the distribution of scattering-length density in 38% D 2 0 (orange) and 70% D 0 (blue) close to the three-fold axis of TBSV. The chain trace of three C-type subunits meeting in a -annulus centred on the three-fold symmetry axis is also indicated (in white). The view is taken approximately perpendicular to the three-fold axis.

5 RNA and protein in tomato bushy stunt virus Timmins, Wild and Witz 1195 residues, apart from Arg98, of the segment immediately preceding the IPB strand of the A and B subunits possess a high contrast in 70% D 2 0 buffer; this holds true especially for the segment K(92)PKFT(96), as well as for Thr99. This implies that the polypeptide that links the R and S domains of the A and B subunits is extended and disordered to such an extent as to make it indistinguishable from solvent. Altogether, the A and B subunits contribute some 90% of the basic residues in the inner shell. The ordered part of the protein subunits clearly contains several regions that possess a positive density in 38% D 2 0 and these regions show up in the corresponding map. They correspond to clusters of hydrophilic amino acids and also show up in the '38% model map' (see the Materials and methods section). One such region corresponds to the loop connecting the B and C strands of the S domains. Others, which become visible in maps drawn at lower density levels, correspond to the interface of the Fig. 5. Superposition of the trace of the C, chains of the three subunits that constitute an icosahedral asymmetric unit of the shell of TBSV, and of the scattering-length density in 38% D 2 0 (red), contoured at a fairly high density. Colour code: subunit A, yellow; subunit B, blue; subunit C, green. A second C subunit from an adjacent, two-fold-related asymmetric unit, is shown in a darker shade of green. (C, chains have been drawn and the maps have been contoured using the software AVS.) P domains making a dimeric projection, or are close to the hinge connecting the S and P domains of a subunit. RNA structure and RNA-protein interactions Fig. 5 shows the 38% D 2 0 map superimposed on the trace of the ordered C a chains of the three protein subunits forming an icosahedral asymmetric unit of the shell (plus the trace of the second C-type subunit contributing to the C-C dimer centred at the two-fold axis). This figure shows that the highest density features of the 38% D 2 0 map that are close to the ordered parts of the protein subunits are located mostly under the C-C dimer, and much less under the A-type and B-type subunits. Furthermore, no appreciable RNA density is evident close to the P-annuli centred at the three-fold axis. Details corresponding to A-type and B-type subunits are shown in Fig. 6, and those corresponding to subunit C in Fig. 7. It is striking that not all basic side chains of the inner surface of the S domains of the capsid come close to patches of RNA density. This holds true especially for residues Lys220 and Lys232 of subunits A and B and for residue Arg221 of subunit A, which definitely point away from a piece of RNA density, although the main chain comes close to it (Fig. 6). It is also striking that Arg86, the first basic residue of the ordered arm of the C-type subunit, does not come close to any feature of significant contrast in 38% D 2 0. Furthermore, Lys202, in strand ef, does not seem to come close to a dense feature of the 38% map in any of the three classes of subunits. All these patches of density that appear in the 38% D 2 0 map may be unambiguously assigned to RNA because they face basic amino acids at the internal surface of the S domains. The interpretation of density features that are not in close contact with side chains ordered in high-resolution maps is more speculative. It is striking that the most visible connection between the outer and inner protein shells, at the icosahedral three-fold axis, is not surrounded by appreciable density in 38% D 2 0, i.e. by matter that could correspond to RNA, although it contains many basic and amino side chains. However, Fig. 4 shows that at smaller radius dense features in both 70% D 2 0 and 38% D 2 0. maps alternate with each other. The protrusions of the inner protein shell, which alternate with dense features in the 38% D2O0 map around the Fig. 6. Stereoview of the details of the possible RNA-protein interactions under A-type and B-type protein subunits of TBSV. The side chains of the basic amino acids of the coat protein are displayed, together with the contours of the 38% D 2 0 map.

6 1196 Structure 1994, Vol 2 No 12 Fig. 7. Stereoview of the details of possible RNA-protein interactions under the C-type subunit of TBSV. Display as for Fig. 6. three-fold axis, make possible close contacts between the RNA and the numerous basic residues of region Lys50- -Lys61, especially as the segment Arg57-Arg60 is probably included in one of the dense regions of the 38% map. No such well-defined features are visible under the A-B type morphological units. Most of the RNA-protein interactions involving the corresponding residues must be confined to the inner protein shell, as these types of subunit contribute >85% of the basic residues of this shell (see above). Discussion Overall structure of the virus In previous studies, with the exception of bean pod mottle virus (BPMV), the very amino-terminal amino acids could not be seen in icosahedrally averaged highresolution maps. In only a very few examples could a significant part of the nucleic acid be located in icosahedrally averaged high-resolution maps: phage )X174 [15], canine parvovirus [16] and flock house virus [17]; BPMV [18] and satellite tobacco mosaic virus (STMV; [19]). In all these viruses, the ordered part of the nucleic acid is nestled in cavities on the interior surface of the capsid. The actual occupancy of the 60 icosahedrally equivalent sites has however not been estimated in the hitherto published structures, with the exception of STMV (full or nearly full occupancy [20]) and phage (X174 (one site in five [15]). In a few other viruses, density inside the ordered part of the protein shell could be visualized at low resolution, but details were lost at high resolution. One such example is southern bean mosaic virus (SBMV): part of the RNA could be located at 5 A resolution [20], but no more detail at 2.8 A [21]. Modelling showed however that a stretch of double-helical RNA could be neatly docked against the ordered basic amino acids facing the interior of the shell, giving rise to other plausible hydrogen bonds [22]. In STNV, most of the RNA could also be located in electron-density maps which included lowresolution terms of the structure factors, because most of it appears to be well separated from the protein by a layer of solvent [9,23]. In TBSV, features were also seen at 16 A resolution [11], but were lost at high resolution [1,2]. Furthermore, protein and RNA cannot be distinguished in these 16 A maps. Comparison of Fig. 6 of [11] with our Figs 1 and 2 shows that, for example, the density maximum located at A radius on the icosahedral three-fold axis clearly corresponds to protein: it corresponds to the connection between the outer and inner protein shells. On the other hand, the density maxima on the five-fold axis and on either side of the threefold axis in Fig. 6 of [11] probably correspond to RNA. STNV constitutes the only other example of a virus that has been studied by low-resolution neutron crystallography using contrast variation [9]. STNV possesses a T=l1 type capsid, and only the 11 amino-terminal amino acids of each protein and all the RNA, are missing in the highresolution electron-density maps. The low-resolution neutron-diffraction study showed that a large part of the RNA is located in rod-shaped features connecting the five-fold vertices of the icosahedron. Three such rods surround the ct-helical coiled coils made up by three subunits meeting along the icosahedral three-fold axis, and can interact with their basic amino acid side chains. Twelve other globular lumps of RNA penetrate into the morphological units, each composed of five subunits. That this structuring is better defined in STNV than in TBSV could well be because in STNV the ordered part of the polypeptide chains comprises 93% of the protein and is sufficient to impose its icosahedral symmetry on the RNA, at least in part. The situation is completely different in TBSV. Because it is a T=3 virus, the three icosahedral classes of protein subunits do not interact with the RNA in the same way. Furthermore, the relative number of amino acids that are not seen in high-resolution maps is much greater than in STNV, and these regions contain many basic amino acids (15 arginine and lysine residues in chains A and B, and 11 such residues in chains C), as well as many amine residues, probably giving rise to very strong RNA-protein interactions. Indeed, the structure of TBSV described in this paper confirms qualitatively the neutron solution scattering results of Chauvin et al. [10], who described the structure in terms of four alternating concentric shells of protein and RNA, with most of the RNA sandwiched between two protein shells, one corresponding to the S and P domains, and the other to most of the R domains. We have extended those results by showing how the

7 RNA and protein in tomato bushy stunt virus Timmins, Wild and Witz 1197 density is localized within these regions, whose radial extents and scattering densities vary widely with the azimuthal position. Another important feature is the existence of 'lakes' of solvent, within both the RNA-containing and the protein-containing regions. The largest of the lakes is located in the intermediate shell which, according to solution studies, accommodates most of the viral RNA, with an average packing density of % (v/v). This suggests that in regions of high RNA density the nucleotides are in fact very tightly packed. Quasi-equivalence and the internal organization of TBSV High-resolution diffraction studies have shown that packing of more than 60 protein molecules in a capsid is achieved by using several different contact conformations (two in a T=3 shell; reviewed in [4,5]), although the underlying icosahedral lattices are those predicted by Caspar and Klug's theory of quasi-equivalence (discussed in [24]). These basic principles do not apply to the RNA, because it does not possess any symmetry in the distribution of its nucleotides. Our results show that in TBSV, connections between the two protein shells indeed take place mainly around the icosahedral threefold axis of the virion. Structural differences between the classes of coat-protein subunits become very important in the interior of the virion, where quasi-equivalence breaks down completely. Fig. 4, and even more strikingly Figs 5 and 6, clearly show that, as far as possible where RNA-protein interactions are concerned, subunits of class C differ much more from those of classes A or B than could be concluded from the high-resolution model of the capsid [1,2]. A similar conclusion holds for the partitioning of the polypeptide chains between the outer and inner protein shells since protein density becomes very weak immediately after the last ordered residues of the A-type and B-type S domains, whereas many amino acids of the C-type subunits are involved in the connections between the two shells. The non-equivalent interactions of the protein subunits with the viral RNA therefore have profound influences on their positioning (and vice versa). Assembly of T=3 viruses Two models have been proposed to account for the morphogenesis of a T=3 shell made of three quasiequivalent classes of the same subunit. The strength and large number of dimeric and five-fold intersubunit contacts in southern bean mosaic virus (SBMV) led Rossmann et al. [25] to suggest that self-assembly of the capsid starts with the formation of an initial 10-mer consisting of five A-B dimers. Further dimers would bind in the 'tensed' C-C state to this decamer, followed by further dimers in the A-B and C-C conformations, etc. In a detailed study of the re-association of turnip crinkle virus (TCV) from dissociated protein dimers and RNA, Sorger et al. [26] noticed that the first nucleoprotein complex to be formed consists of the viral RNA and approximately six protein subunits. They suggested, therefore, that selfassembly starts with the formation of a set of three C-C dimers connected by a three-stranded -annulus upon binding to the RNA. The next subunit dimers to bind would have to adopt an A-B conformation because steric hindrance prevents the arm from ordering [27]. Initial stages of the assembly may involve specific sites and protein, as well as RNA, conformations that could occur at only one place in a virion and differ considerably from the average structure. These would of course be masked by the averaging procedures used to calculate our density maps as well as the high-resolution ones: for example, the single copy of A protein present in phage MS2 is not visible in Liljas' model (discussed in [7]). If these structural singularities are not too important, our model of TBSV may provide some hints about the assembly of TBSV. Our model further emphasizes the differences between A-B and C-C dimers that were already apparent from high-resolution studies. From this point of view it could support either of the two existing models of assembly. However, interaction of an initial 10-mer of A-B dimers would imply that most of the interactions of this 'nucleus' with the viral RNA would be located more than 30 A away from the inner surface of the S domains, connected to this surface by only tenuous protein stretches extending through lakes of solvent. Our model agrees much better with the 'three C-C dimers first' model, as RNA-protein interactions with the C subunits are much stronger than with the A or B classes, and as the only visible connection between the outer and inner protein shells takes place at the three-fold axis, where three C subunits meet in a 13-annulus. Biological implications Tomato bushy stunt virus (TBSV) is a typical example of a small isometric virus for which a high-resolution model of the capsid has been published. Its T=3 protein shell is made up of 180 copies of a single protein species, arranged in three different conformations (A, B, C). All of the viral RNA as well as -25% of the protein (corresponding to the amino termini of the polypeptide chains) are missing in the high-resolution model because they do not possess icosahedral symmetry. However, the missing parts of the structure play a very important role in the assembly and stability of the virions because they contain many basic amino acids able to interact with the RNA. Solution studies have shown that most of this 'missing protein' forms a second, internal shell, and that most of the RNA is sandwiched between the two protein shells and rigidly linked to the capsid. In order to gain insight into the organization of the interior of the virion we have measured neutron-diffraction data from single crystals of TBSV in solvents of various H 2 0/D 2 0 molar ratios, at a resolution of 16 A. Neutrons are scattered by the nuclei of atoms and the scattering of mixtures of H 2 0 and D 2 0 varies considerably with the D 2 0 content of the buffer. At low resolution, the

8 1198 Structure 1994, Vol 2 No 12 average contrast of the protein is zero in 38% D 2 0, and that of RNA is zero in 70% D 2 0. Using the high-resolution model as a starting model, we were able to interpret the neutron-diffraction data and to locate in three dimensions those regions where RNA or protein accumulate in the virion. Our results confirm that most of the protein is distributed among two shells, which 'sandwich' the RNA, but the distribution of density in the inner shells is very inhomogeneous, to an extent that could not be predicted from the high-resolution model. In that model, which corresponds to the outer shell of our density maps, A-type or B- type subunits differ from C-type ones, mainly at the level of intersubunit contacts, because a 35-residue long arm is ordered only in the C subunits. We show that these structural differences become considerably more important in the interior of the virion. Particularly striking are differences in their interactions with the viral RNA which allow the classes of chemically identical coat protein subunits to play very different roles in the stability, and therefore also probably in the assembly and disassembly, of the virions. These differences make simple T=3 viruses look much more like picornaviruses than could be expected from the quasi-equivalence rules. Materials and methods Sample preparation TBSV was propagated in Datura stramonium. Plant proteins were removed from the sap by precipitation at ph 4.8, and the virus was purified by two or three cycles of high-speed and low-speed centrifugation. Virus solutions were filtered through 0.22 pim Millex filters. Crystals (up to mm in size) were grown at 4 0 C in ammonium sulphate, as described by Harrison and Jack [11]. They were transferred into 0.9 M sodium sulphate at room temperature, and soaked for several weeks in several changes of the same saline solution containing the appropriate amount of D 2 0. For data collection, crystals were mounted in mm diameter quartz capillaries containing a drop of the last mother liquor. The D 2 0 content of this mother liquor was estimated by measuring the neutron transmission coefficient of an aliquot contained in a 1 mm or 2 mm Hellma quartz cuvette, using the Institut Laue-Langevin (ILL) small-angle scattering instrument D 11 [28]. The space group of the crystals is 123 (a=386 A), one particle being associated with each lattice point of the body-centred cell. The crystallographic asymmetric unit contains 1/12 of a virion but, as pointed out in the Introduction section, crystal packing leads to an icosahedral averaging of all structural details. Data collection Data from single crystals of TBSV soaked in solvents of various H 2 0/D 2 0 molar ratios were measured on the D17 small-angle scattering instrument at the ILL, Grenoble. The wavelength (X) was 10.8 A, AX/X=5% (full width half maximum) and was selected from the white beam by a helical slot velocity selector. The beam divergence was 1. A 64 cmx64 cm BF 3 area detector with pixel size of 1 cmx1 cm was used for data collection with normal beam geometry and a crystal-to-detector distance of 80 cm. The data were measured to 16 A resolution for 0%, 70% and 100% D 2 0 in H20, and to 29 A for 38% D20/H20, using -scans of per step (data corresponding to 38% D 2 0 were recorded at a lower resolution because crystals scatter only weakly at this contrast). Data collection times varied typically between 3 and 20 days depending on the contrast. Data reduction Data were reduced using a suite of programs developed by Roth et al. [29,30]. Integration was carried out using an analytical expression for the intensity masks. No correction for the beam absorption was applied. Symmetry merging R-factors for the four data sets varied between 5% and 10% (see Table 1). The method of low-resolution neutron crystallography by contrast variation depends on the fact that crystals contain a large proportion of water, typically 40-50%. The H 2 0/D 2 0 content of the solvent can be varied, and the structure factors are expressed as a linear function of the D 2 0 mole fraction: F(X,h)=F(O,h) + XFHD(h) (1) where F(O,h) is the structure factor at the reciprocal lattice point h for the structure in H 2 0; FHD(h) is the structure factor arising from the H/D exchange and H 2 0/D 2 0 change; and X is the mole fraction of deuterium in the solvent. The measured intensity of a reflection h, after appropriate scaling, is given by: I(h)= I FH X I FH20 I FHD I cos+x 2 I FHD 2 (2) where q is the phase difference between FH20 and FHD. Thus, the scattered intensity is a parabolic function of the contrast, and this property is used by the programs of Roth et al. [30] to scale together the data sets measured at different contrasts. In the particular case where the phase difference is 0 or rt (e.g. for centrosymmetric reflections), the structure factor amplitude becomes a linear function of contrast. This procedure is exactly analogous to the single isomorphous replacement technique in X-ray protein crystallography, and as such can be used to transfer phase information from one contrast to another. If the structure of the particle at one contrast is known (e.g. the unknown component is contrast-matched), then the phase at a contrast where the structure is not known may be calculated with an ambiguity of sign (see Fig. 1 of [9]). If the structure amplitude at two contrasts is known, then that at any other contrast may be calculated without ambiguity. In space group 123, reflections of the type hkl and khl are not equivalent and it was therefore necessary to ensure that the correct choice of axes was made for each contrast. This was carried out by first scaling together the data sets using only reflections of the type hho, where this ambiguity does not exist, and then examining whether an indexing of the type hkl or khl gave the best fit to the scaled amplitudes. The scaling program also allows for the refinement of the D 2 0 concentrations, but only where a minimum of four contrasts have been measured. As our data from all contrasts scaled together consistently using the D0,O concentrations determined from transmission measurements of the mother liquors, it was unnecessary to further refine these values. Unmeasured data for the 38% D 2 0 contrast were interpolated according to equation (2) and the phase difference for each contrast was calculated. The reliability of the interpolated data

9 RNA and protein in tomato bushy stunt virus Timmins, Wild and Witz 1199 Table 1. Data collection statistics. D 2 0 content Total no. of No. of unique No. of unique Completeness Rsym (> 3) (%0/) measured measured maesured of data (%) (%) reflections reflections reflections > 3a a adata set measured only to 29 A resolution. depends on the accuracy of the data in the contrasts which are used for interpolation [30], and which for TBSV are described in Table 1. Non-crystallographic symmetry phase refinement The 60-fold icosahedral symmetry of the TBSV particle was utilized in phase refinement using the procedure described by Bricogne [31,32] and as applied to the high-resolution X-ray structure [2]. Starting phases for the refinement procedure were obtained from the atomic coordinates of the TBSV protein shell as published by Olson et al. [2]. Neutron-scattering factors appropriate to each amino acid were calculated in the following manner. Excess scattering lengths for each amino acid were calculated according to the expression: f(j)=(yb -PsV)H20+X(Fb -psv)hd (3) where Ps is the scattering length density of solvent; lb is the sum of scattering lengths of the atoms of each amino acid; V is the volume of the amino acid residue in H 2 0 (or D 2 0), (see Table in [33]); and X is the mole fraction of deuterium in the solvent. The subscripts H 2 0 and HD refer to the structures in H 2 0 and the difference between H 2 0 and D 2 O, respectively. The excess scattering lengths f(j) were multiplied y a factor exp[(-bsin 2 0)/X 2 ], where B=10 A 2, to provide a form factor corresponding approximately to the Gaussian volume of the residue. Structure factors were then calculated using the atomic coordinates of each C s position in the protein shell and the form factor (calculated as above) for the appropriate amino acid. These 'model' structure factors essentially represent the neutron diffraction expected from the S and P domains of the protein shell of the virus. Fourier maps (Fobs, Otcalc) were then calculated, using the phases of these calculated structure factors and the observed neutron diffraction amplitudes for the 0%, 70% and 100% D 2 0 contrasts. Because, at low resolution, the shape of the virus particle is approximately spherical, the molecular transform of the virus particle is real and can be measured by neutron solution scattering. The signs of the phases of certain low-resolution centrosymmetric reflections could therefore be compared with the neutron solution scattering data [10], and where there was a discrepancy the signs determined from the solution scattering (up to approximately 95 A-') were used. A preliminary map at 38% D 2 0 was calculated using the analogy to the single isomorphous replacement described above. This calculation used structure factor amplitudes of F 70 cosatx, where Aot is the phase difference between F70 and F38, and the mean phase for the F 38, i.e. (x 70. The phases for the 0%, 70% and 100% contrasts were then refined by the method of non-crystallographic symmetry averaging. The procedure used was essentially that described by Olson et al. [2]. In space group 123, with the origin at position (/2,/2,/2) of the unit cell, icosahedral averaging reduces to averaging the map about a single five-fold axis. In this study only one grid (of 3.84 A) was used for the averaging, because of the lower resolution. Solvent flattening was applied only to regions outside the virus particle because density was expected to occur in the interior of the virus. After each cycle of averaging, the density outside the molecular envelope was set to its average. Calculated phases were applied to the observed structure factors and a new map was calculated. The results of these refinements are summarized in Table 2. Density map calculations Following the refinement of the 0%, 70% and 100% contrasts, 'optimal' density maps [34] were calculated using the program PROPHA [35]. This method is based on the use of a joint probability distribution of the measured intensities and calculated structure factors for each reflection (h), and allows the merging of data measured at different contrasts in order to calculate an optimal map at any contrast. The program also permits the interpolation or extrapolation of structure factors from the contrasts for which densities have been determined to another contrast using the structure factor relationship given by equation (2). 'Best' estimates of the structure factor amplitudes and phases were thus obtained for the 0%, 70% and 100% contrasts, and used to calculate Fcalc, Cocalc maps. Interpolated values for the 38% D 2 0 contrast were also obtained from this program. A Fourier map at 38% D 2 0 was then calculated by using these calculated phases and the measured 38% amplitudes (where available) and interpolated structure-factor amplitudes for unmeasured data. The resulting map was then refined by icosahedral averaging as above; the results obtained are summarized in Table 2. The maps at all four contrasts were put onto an absolute scale in the following way. The densities at all points contained within the outer protein shell of the virus, above the average background, were summed in each of the four contrast maps. The envelope used to delimit the shell at 16 A resolution was obtained from a Fourier map calculated with phases and 'model' neutron structure-factor amplitudes for 70% D 2 0 calculated from the published Ca coordinates as described in the previous section. This map (referred to in this paper as the '70% model map') produced a reasonable image of the outer protein shell. Because the amino acid composition of the protein was known, the theoretical neutron excess scattering length (bp 5 V) of the shell could be calculated and the scale factors required to give the same scattering densities in the maps obtained. Once the maps are on the same scale, the linear variation of scattering density (and hence structure factor) with

10 1200 Structure 1994, Vol 2 No 12 Table 2. Progress of refinement. D 2 0 content () R-factor at start (%) R-factor at finish (%) R-factor = hki l Fob, - kfcaic /hki Fob I contrast (equation ) allows the match point of each point in the density map to be calculated. This is the mole fraction of D2O in the solvent at which the scattering caused by the structure is matched by that of the solvent. It is thus given by the abscissa intercept of a plot of scattering density against percentage D 2 0. A map of'match-points' was calculated in this manner. Interpretation of density maps The average contributions of TBSV coat protein and RNA at each contrast are given in Fig. 8, together with the contributions of individual amino acids. On average, only RNA contributes to the scattering in 38% DO 2 0, whereas the diffraction pattern observed in 70% D 2 0 is attributable to the protein moiety of the virions. Inhomogeneities within the RNA (i.e. differences in scattering-length densities between sugars, bases and phosphates) should be insignificant at the experimental resolution used. On the other hand, local fluctuations caused by particular amino acids may be important. Hydrophilic residues such as aspartate, asparagine, histidine or arginine, for example, possess a contrast in 38% D 2 0 that equals half of that of compact RNA. Stretches of the polypeptide chain rich in 38 D 2 0 (%, molar ratio) Fig. 8. The contributions of individual amino acids to the scattering (p-p ) as a function of H 2 0/D 2 0 content of the solvent. The diagonal line represents the scattering length density of the solvent. The scattering length densities of the individual amino acids are shown on the four vertical lines representing the experimental contrasts. The contrast for each amino acid is therefore the difference between its scattering length density at a given H 2 0/D 2 0 content of the solvent and that of the solvent itself. Amino acids are represented by the standard one-letter code. The dotted line represents the average scattering length densities of TBSV protein. 0.7 U~ x l such residues may therefore show up in a 16 A resolution density map at this contrast. We have checked that this is indeed the case in the '38% model map' calculated in the same way as the '70% model map', but corresponding to the contrast in 38% DO instead of 70%. Clusters of aliphatic residues such as valine, methionine, leucine and isoleucine possess a strong negative contrast in 38% D2O. In 70% D2O the contribution of all residues is negative. Arginine, asparagine, aspartate and serine possess only very weak contrast, whereas the contributions of proline, valine, methionine, leucine and isoleucine are very important. The five-fold symmetry axes of the icosahedron are skew with respect to the coordinate axes. This results in details of the maps being slightly different from one subunit to another, because of sampling problems [36], even where the maps have been icosahedrally averaged. These differences become more important at small radii, as the number of sampling points comprised between any given symmetry axes of an isometric object decreases. Essential features remain equivalent, although they may correspond to different density levels for different subunits. We have checked that all features described in this paper hold for all five symmetry-related subunits, although only one may have been used for the illustrations. Another question that arises in the refinement of such structures at low resolution is the identification of criteria for judging the correctness of the final structure. At high-resolution one has the constraints of atomic-resolution molecular geometry from known amino acid conformations and dimensions. At low resolution no such constraints are available. In the case of TBSV, however, we do have a known partial structure: the protein outer coat. The fact that after independent refinement of the whole structure at four different contrasts the structure of the known part is faithfully preserved, both in terms of density location and its variation with contrast, gives us confidence in the correctness of the procedures. Acknoivledgements: We wish to thank Professor Steve Harrison for numerous discussions and insights into TBSV structure throughout the course of this project. We are also grateful to Dr Michel Roth for help in the use of his programs and to Dr Bernard Jacrot who suggested the problem and for his constant encouragement. One of us (W) was in part supported by a short-term EMBO fellowship. References 1. 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