( A ) 8.5 A Ê resolution projection map calculated from eight averaged images of glucose-embedded orthorhombic crystals, assuming p 22 1 2 1 symmetry. Exact circles have been drawn through the innermost 

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... form . A representation of all the Fourier components obtained from one image is shown in Figure 3A. An analysis of symmetry-related phases showed the data to be consistent with p 42 1 2 plane group symmetry (Valpuesta et al ., 1994). Amplitudes and phases from the ®ve best images were merged and averaged, imposing either p 1 or p 42 1 2 symmetry with a = b = 165 A Ê. Origin and phase contrast transfer function (CTF) re®nement were performed independently for the two plane groups, with Fourier terms limited to 8.5 A Ê resolution. Table I shows the average phase residual as a function of resolution for p 42 2 symmetry, indicating non-random data to 8.5 A Ê resolution. Amplitudes from individual images were corrected by temperature factors of between 172 and 492 A Ê 2 prior to averaging. The projection map with p 1 symmetry (Figure 3B) reveals the a -helical structure of the LH1 antenna, showing both `up' and `down' orientations of the complex. One orientation displays higher contrast than the other. This re ̄ects some differences in penetration of the embedding medium between the side interacting with the carbon support and the side interacting with the air, affecting the overall contrast of the molecular envelope. If lower order terms are excluded from the Fourier synthesis, the contrast appears similar for the two orientations (data not shown). Each complex can be seen to be composed of a central mass (the RC) enclosed by a 16-subunit LH1 ring. The LH1 ring has a projection structure similar to that reported by Karrasch et al . (1995) for LH1 crystals embedded in ice. Figure 3C shows the equivalent map with p 42 1 2 symmetry averaging applied and the central RC density clearly contrasted against the background. The 16-fold symmetry of the LH1 complex is clearly evident. Whilst the central RC density has an imposed 4-fold symmetry, the RC density at a high radial distance from the 4-fold origin gives some indication of even higher symmetry. Previous analysis has shown that there could only be room for one RC within the complex (Karrasch et al ., 1995), and so any symmetry in the RC density must re ̄ect the superposition of single RCs in different orientations over all the complexes imaged (Stahlberg et al ., 1998; Walz et al ., 1998). Analysis of rotational symmetry in tetragonal form . Although the observed image phases were consistent with p 42 1 2 symmetry, we considered the possibility that this only re ̄ected the symmetry arising from the highly contrasted 16-fold LH1 rings related by crystallographic screw axes in the crystal plane. As a test of the crystallographic symmetry of the RCs, we initially merged and re®ned the data from individual images assuming no crystallographic symmetry. Figure 4A shows the rotational power spectrum for the central density of one of the two crystallographically independent complexes of the p 1 projection (Figure 3B) out to a radius of 28 A Ê (thereby excluding any LH1 density) (Crowther and Amos, 1971). This indicates a signi®cant 4-fold rotational symmetry. Figure 4B shows a 4-fold rotationally ®ltered view of the complex in which four lobes of density are visible, projecting from the centre. We computed the density correlation coef®cients between the two crystallographically independent RC densities of the p 1 projection (after 4-fold rotational averaging of the density) within the program suite IMAGIC (van Heel et al ., 1996). Comparison of densities out to a radius of 28 A Ê gave the highest cross-correlation coef®cient (55%) when the mirror image of one RC was matched with the unre ̄ected image of the other RC, without any rotation, and a translation of less than one pixel. This is consistent with the RC densities being related by an in-plane 2-fold screw axis independently of the LH1 densities. In other words, the RC density obeys the crystallographic p 42 1 2 symmetry. The rotational power spectrum of the p 42 1 2 averaged RC±LH1 complex is dominated by the 16-fold symmetry of the outer LH1 ring (Figure 4C). Interestingly, a rotational power spectrum of a ring of density between 22 and 39 A Ê radius (Figure 4D) (i.e. not including any LH1 density) shows 8-, 12- and 16-fold harmonics, with the 16-fold being relatively strong. This indicates a possible 16-fold symmetry for the RC density in addition to the crystallographic 4-fold symmetry. Orthorhombic form . An analysis of symmetry-related phases showed the data to be consistent with p 22 1 2 1 plane group symmetry (Valpuesta et al ., 1994). Amplitudes and phases from the eight best images were merged and averaged, imposing either p 1 or p 22 1 2 1 symmetry. Origin and CTF re®nement was performed independently for the two plane groups, with Fourier terms limited to 8.5 A Ê resolution. Table I shows the average phase residual as a function of resolution for p 22 1 2 1 symmetry, indicating non-random data to 8.5 A Ê resolution. Again we see two oppositely oriented RC±LH1 complexes, with a 16- subunit LH1 ring (Figure 5). The central RC density, which has imposed 2-fold symmetry, does not show any marked higher symmetry in the way that the RC in the tetragonal crystal form does, suggesting a preference for only two orientations relative to the crystal lattice, with the consequent 2-fold averaging of the central density. In this crystal form, the negative staining pattern does not give the impression of a square RC±LH1 complex (Figure 2C), but, remarkably, the surrounding LH1 is also not circular. Figure 5A shows how the density peak positions for the putative a -helices of the subunits deviate from an exact circular assembly. These peak positions describe an ellipse whose major and minor axes differ by ~11%. The 8.5 A Ê resolution projection structures of the R.rubrum RC±LH1 complex (Figures 3C and 5) are suf®ciently detailed to clearly show 16 LH1 ab subunits arranged in a closed ring around the central RC density. The rotational power spectrum of the tetragonal form (Figure 4C) shows an unambiguous 16-fold symmetry; no signi®cant differ- ence can be observed between the densities representing the crystallographically independent LH1 subunits, lead- ing to the conclusion that the RC is surrounded by 16 identical protein subunits. There is no evidence in these projection maps for any other minor protein component in the outer LH1 ring. In both maps, the area enclosed within the LH1 ring is occupied by signi®cant positive density (overall mean unit cell density is zero); this density corresponds to the RC, as proposed by earlier workers for RC±LH1 complexes from a number of species (Miller, 1982; Stark et al ., 1984; Engelhardt et al ., 1986; Boonstra et al ., 1994; Karrasch et al ., 1995; Walz and Ghosh, 1997; Stahlberg et al ., 1998; Walz et al ., 1998). Although the LH1 ring is only able to accommodate one RC, the RC density apparently has a signi®cant 4-fold rotational symmetry in the tetragonal form and 2-fold symmetry in the orthorhombic form. By treating RC±LH1 complexes in negatively stained tetragonal 2D crystals as single par- ticles, Stahlberg et al . (1998) showed that individual RCs adopted one of four possible orientations with respect to the crystal axes; the 4-fold symmetry we see in our analysis simply arises from the superposition of all RCs in these four possible orientations. However, the rotational power spectrum of the RC density at higher radius, in addition to showing a 4-fold component, shows an even stronger 16-fold harmonic (Figure 4D), suggesting that there must also be a 16-fold symmetry to the RC density, not resolved at lower radius. The propensity of the RC to adopt up to 16 discrete orientations suggests a speci®c interaction binding the RC within the antenna ring. It is likely that the crystal contacts perturb the 16-fold symmetry of the LH1 so as to favour four major occupancy RC orientations, with a further 12 minor occupancy orientations to give 16 in total. An analysis for the orthorhombic form indicated only two major orientations for the RC and very low occupancy indeed of other minor orientations (data not shown). What the structure of this orthorhombic form clearly demon- strates is the ability of the LH1 ring to adopt circular and elliptical conformations. Inspection of the RC complex in projection shows that it possesses long and short axes, so it might be expected that the LH1 ring could distort to pack round the RC, when subjected to external packing constraints in the orthorhombic crystal lattice. The 8.5 A Ê projection data for LH1 from R.rubrum (Karrasch et al ., 1995), together with the present data on RC±LH1, provide a valuable opportunity to assess the effect of binding one membrane protein to another, in this case the association of a RC with a light-harvesting complex. It should be noted that there was a difference in the way that the two complexes, LH1 and RC±LH1, were prepared. In the study of Karrasch et al ., the 2D crystals had been grown from dissociated subunits prepared from a carotenoidless mutant of R.rubrum ; in the present study, the RC±LH1 complex contained carotenoids, and was not dissociated at any step of the puri®cation or crystallization, so it is likely that the 16-fold arrangement of subunits represents the in vivo structure. Despite this difference between LH1 and RC±LH1 complexes in terms of preparation, the peaks of density of the a - and b -chains have, within experimental uncertainty, the same coordinates in both the LH1 map of Karrasch et al . (1995) and our tetragonal map (Figure 3C), suggesting that there has been no signi®cant conformational change within the individual subunits upon incorporation of the RC. However, the elliptical arrangement of LH1 subunits in the orthorhombic crystal form (Figure 5) shows that a conformational change in the entire LH1 assembly is possible. This points to a ̄exibility in the whole LH1 ring assembly, which may lead to ̄uctuations in the degree of ellipticity, as has been inferred from ...

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... A Ê resolution. Table I shows the average phase residual as a function of resolution for p 22 1 2 1 symmetry, indicating non-random data to 8.5 A Ê resolution. Again we see two oppositely oriented RC±LH1 complexes, with a 16- subunit LH1 ring (Figure 5). The central RC density, which has imposed 2-fold symmetry, does not show any marked higher symmetry in the way that the RC in the tetragonal crystal form does, suggesting a preference for only two orientations relative to the crystal lattice, with the consequent 2-fold averaging of the central density. In this crystal form, the negative staining pattern does not give the impression of a square RC±LH1 complex (Figure 2C), but, remarkably, the surrounding LH1 is also not circular. Figure 5A shows how the density peak positions for the putative a -helices of the subunits deviate from an exact circular assembly. These peak positions describe an ellipse whose major and minor axes differ by ~11%. The 8.5 A Ê resolution projection structures of the R.rubrum RC±LH1 complex (Figures 3C and 5) are suf®ciently detailed to clearly show 16 LH1 ab subunits arranged in a closed ring around the central RC density. The rotational power spectrum of the tetragonal form (Figure 4C) shows an unambiguous 16-fold symmetry; no signi®cant differ- ence can be observed between the densities representing the crystallographically independent LH1 subunits, lead- ing to the conclusion that the RC is surrounded by 16 identical protein subunits. There is no evidence in these projection maps for any other minor protein component in the outer LH1 ring. In both maps, the area enclosed within the LH1 ring is occupied by signi®cant positive density (overall mean unit cell density is zero); this density corresponds to the RC, as proposed by earlier workers for RC±LH1 complexes from a number of species (Miller, 1982; Stark et al ., 1984; Engelhardt et al ., 1986; Boonstra et al ., 1994; Karrasch et al ., 1995; Walz and Ghosh, 1997; Stahlberg et al ., 1998; Walz et al ., 1998). Although the LH1 ring is only able to accommodate one RC, the RC density apparently has a signi®cant 4-fold rotational symmetry in the tetragonal form and 2-fold symmetry in the orthorhombic form. By treating RC±LH1 complexes in negatively stained tetragonal 2D crystals as single par- ticles, Stahlberg et al . (1998) showed that individual RCs adopted one of four possible orientations with respect to the crystal axes; the 4-fold symmetry we see in our analysis simply arises from the superposition of all RCs in these four possible orientations. However, the rotational power spectrum of the RC density at higher radius, in addition to showing a 4-fold component, shows an even stronger 16-fold harmonic (Figure 4D), suggesting that there must also be a 16-fold symmetry to the RC density, not resolved at lower radius. The propensity of the RC to adopt up to 16 discrete orientations suggests a speci®c interaction binding the RC within the antenna ring. It is likely that the crystal contacts perturb the 16-fold symmetry of the LH1 so as to favour four major occupancy RC orientations, with a further 12 minor occupancy orientations to give 16 in total. An analysis for the orthorhombic form indicated only two major orientations for the RC and very low occupancy indeed of other minor orientations (data not shown). What the structure of this orthorhombic form clearly demon- strates is the ability of the LH1 ring to adopt circular and elliptical conformations. Inspection of the RC complex in projection shows that it possesses long and short axes, so it might be expected that the LH1 ring could distort to pack round the RC, when subjected to external packing constraints in the orthorhombic crystal lattice. The 8.5 A Ê projection data for LH1 from R.rubrum (Karrasch et al ., 1995), together with the present data on RC±LH1, provide a valuable opportunity to assess the effect of binding one membrane protein to another, in this case the association of a RC with a light-harvesting complex. It should be noted that there was a difference in the way that the two complexes, LH1 and RC±LH1, were prepared. In the study of Karrasch et al ., the 2D crystals had been grown from dissociated subunits prepared from a carotenoidless mutant of R.rubrum ; in the present study, the RC±LH1 complex contained carotenoids, and was not dissociated at any step of the puri®cation or crystallization, so it is likely that the 16-fold arrangement of subunits represents the in vivo structure. Despite this difference between LH1 and RC±LH1 complexes in terms of preparation, the peaks of density of the a - and b -chains have, within experimental uncertainty, the same coordinates in both the LH1 map of Karrasch et al . (1995) and our tetragonal map (Figure 3C), suggesting that there has been no signi®cant conformational change within the individual subunits upon incorporation of the RC. However, the elliptical arrangement of LH1 subunits in the orthorhombic crystal form (Figure 5) shows that a conformational change in the entire LH1 assembly is possible. This points to a ̄exibility in the whole LH1 ring assembly, which may lead to ̄uctuations in the degree of ellipticity, as has been inferred from ̄uorescence studies of LH2 complexes immobilized on a mica surface (Bopp et al ., 1999). Our p 42 1 2 crystals display the same characteristics as those of a carotenoidless mutant analysed at lower resolution by Walz and Ghosh (1997) and Stahlberg et al . (1998), with similar unit cell dimensions and four high- occupancy orientations for the RC with respect to the crystal axes. Moreover, maps from crystals embedded in negative stain (Figure 2A) or negative stain±glucose mixtures (Figure 2B) can give a rather `square' appearance to the RC±LH1 complexes, depending on the degree of staining. However, negative stain tends to contrast mainly the external surfaces of 2D crystals, and this square-like appearance most likely arises from the accumulation of stain in `pools' arranged in the square crystal lattice. In our images of unstained crystals where the internal protein structure is revealed, the LH1 appears to show little deviation from a true circle in the tetragonal crystal form (Figure 3C). There is no evidence of any square-like distortion of the LH1 induced by either an additional protein component or by crystal packing, as had been suggested by earlier work (Stahlberg et al ., 1998). Moreover, we have discovered a new orthorhombic crystal form, which when embedded in negative stain always appears to contain `circular' complexes (Figure 2C). Subsequent higher resolution analysis does indicate a signi®cant deviation from circular symmetry, but into a 2-fold symmetric elliptical form, not as a square (Figure 5). We have attempted, in an approximate way, to model this 2-fold symmetric RC density using atomic coordinates derived from the R.sphaeroides structure. We have assumed that contrast will arise mainly from protein embedded within the transmembrane region, but rather than attempt to model the density of the surrounding medium, we have calculated an 8.5 A Ê projection map from coordinates of atoms in a vacuum. A projection perpendicular to the putative membrane plane was calculated. Rotational and translational alignment of this density, followed by 2-fold averaging, gave a correlation coef®cient between model and experimental density of 0.7 for the best ®t. In this orientation, the four RC light subunit helices L A , L B , L C and L E are superimposed on four relatively dense regions separated by ~10 A Ê in the RC map (marked in red in Figure 6A). The long axis of the RC model coincides approximately with the major axis of the elliptical LH1 ring. The pseudo 2-fold axis of the RC running through the Fe and between the `special pair' of Bchls lies within 2±3 A Ê of the crystallographic 2-fold axis. With the uncertainties that arise from calculating model density at low resolution in such a complex environment of lipid, protein, pigment and glucose, the uncertainty in orientation must be at least T 3 ° . However, within this degree of uncertainty, the planes of the Bchl porphyrin rings make an angle of ~43 ° with the major axis of the LH1 ring. There are few data on the detailed nature of the interactions of the RC with the LH1, although cross- linking experiments suggest that L, M and H subunits all interact in some way with LH1 subunits (Peters et al ., 1983). In our model, within the membrane, the only RC transmembrane helices that could interact with the putative LH1 a helices would be L A , M A and possibly L B and M B ; all other helices are much further than 10 A Ê from their nearest LH1 neighbours. A near-straight line can be drawn linking the centres of gravity of two diametrically opposed LH1 a subunits and the L and M helices. This would be consistent with a helix±helix packing arrangement of L A and M A against nearest-neighbour LH1 a subunits. This study represents the highest resolution view, to date, of any RC±LH1 complex from a purple bacterium. The previous study by Karrasch et al . (1995), which indicated that R.rubrum forms a closed LH1 ring, raised the question of how quinone transfer takes place between the apparently fully encircled RC and the spatially distant cytochrome bc 1 complex during cyclic electron ̄ow. The projection maps of the RC±LH1 complex (Figures 3C and 5) would appear to con®rm that the RC is indeed completely enclosed by 16 identical LH1 subunits, so that the mechanism of quinone transfer remains unclear. This is in the context of work on the PufX polypeptide from R.sphaeroides and Rhodobacter capsulatus , which has shown that it facilitates the transport of reducing equivalents from the RC to the bc 1 complex (Farchaus et al ., 1990, 1992; Lilburn et al ., 1992). It has been shown that PufX is unnecessary in strains that cannot assemble a normal LH1 complex (McGlynn et al ., 1994, 1996). It has been suggested that PufX might ...

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... RC, as proposed by earlier workers for RC±LH1 complexes from a number of species (Miller, 1982; Stark et al ., 1984; Engelhardt et al ., 1986; Boonstra et al ., 1994; Karrasch et al ., 1995; Walz and Ghosh, 1997; Stahlberg et al ., 1998; Walz et al ., 1998). Although the LH1 ring is only able to accommodate one RC, the RC density apparently has a signi®cant 4-fold rotational symmetry in the tetragonal form and 2-fold symmetry in the orthorhombic form. By treating RC±LH1 complexes in negatively stained tetragonal 2D crystals as single par- ticles, Stahlberg et al . (1998) showed that individual RCs adopted one of four possible orientations with respect to the crystal axes; the 4-fold symmetry we see in our analysis simply arises from the superposition of all RCs in these four possible orientations. However, the rotational power spectrum of the RC density at higher radius, in addition to showing a 4-fold component, shows an even stronger 16-fold harmonic (Figure 4D), suggesting that there must also be a 16-fold symmetry to the RC density, not resolved at lower radius. The propensity of the RC to adopt up to 16 discrete orientations suggests a speci®c interaction binding the RC within the antenna ring. It is likely that the crystal contacts perturb the 16-fold symmetry of the LH1 so as to favour four major occupancy RC orientations, with a further 12 minor occupancy orientations to give 16 in total. An analysis for the orthorhombic form indicated only two major orientations for the RC and very low occupancy indeed of other minor orientations (data not shown). What the structure of this orthorhombic form clearly demon- strates is the ability of the LH1 ring to adopt circular and elliptical conformations. Inspection of the RC complex in projection shows that it possesses long and short axes, so it might be expected that the LH1 ring could distort to pack round the RC, when subjected to external packing constraints in the orthorhombic crystal lattice. The 8.5 A Ê projection data for LH1 from R.rubrum (Karrasch et al ., 1995), together with the present data on RC±LH1, provide a valuable opportunity to assess the effect of binding one membrane protein to another, in this case the association of a RC with a light-harvesting complex. It should be noted that there was a difference in the way that the two complexes, LH1 and RC±LH1, were prepared. In the study of Karrasch et al ., the 2D crystals had been grown from dissociated subunits prepared from a carotenoidless mutant of R.rubrum ; in the present study, the RC±LH1 complex contained carotenoids, and was not dissociated at any step of the puri®cation or crystallization, so it is likely that the 16-fold arrangement of subunits represents the in vivo structure. Despite this difference between LH1 and RC±LH1 complexes in terms of preparation, the peaks of density of the a - and b -chains have, within experimental uncertainty, the same coordinates in both the LH1 map of Karrasch et al . (1995) and our tetragonal map (Figure 3C), suggesting that there has been no signi®cant conformational change within the individual subunits upon incorporation of the RC. However, the elliptical arrangement of LH1 subunits in the orthorhombic crystal form (Figure 5) shows that a conformational change in the entire LH1 assembly is possible. This points to a ̄exibility in the whole LH1 ring assembly, which may lead to ̄uctuations in the degree of ellipticity, as has been inferred from ̄uorescence studies of LH2 complexes immobilized on a mica surface (Bopp et al ., 1999). Our p 42 1 2 crystals display the same characteristics as those of a carotenoidless mutant analysed at lower resolution by Walz and Ghosh (1997) and Stahlberg et al . (1998), with similar unit cell dimensions and four high- occupancy orientations for the RC with respect to the crystal axes. Moreover, maps from crystals embedded in negative stain (Figure 2A) or negative stain±glucose mixtures (Figure 2B) can give a rather `square' appearance to the RC±LH1 complexes, depending on the degree of staining. However, negative stain tends to contrast mainly the external surfaces of 2D crystals, and this square-like appearance most likely arises from the accumulation of stain in `pools' arranged in the square crystal lattice. In our images of unstained crystals where the internal protein structure is revealed, the LH1 appears to show little deviation from a true circle in the tetragonal crystal form (Figure 3C). There is no evidence of any square-like distortion of the LH1 induced by either an additional protein component or by crystal packing, as had been suggested by earlier work (Stahlberg et al ., 1998). Moreover, we have discovered a new orthorhombic crystal form, which when embedded in negative stain always appears to contain `circular' complexes (Figure 2C). Subsequent higher resolution analysis does indicate a signi®cant deviation from circular symmetry, but into a 2-fold symmetric elliptical form, not as a square (Figure 5). We have attempted, in an approximate way, to model this 2-fold symmetric RC density using atomic coordinates derived from the R.sphaeroides structure. We have assumed that contrast will arise mainly from protein embedded within the transmembrane region, but rather than attempt to model the density of the surrounding medium, we have calculated an 8.5 A Ê projection map from coordinates of atoms in a vacuum. A projection perpendicular to the putative membrane plane was calculated. Rotational and translational alignment of this density, followed by 2-fold averaging, gave a correlation coef®cient between model and experimental density of 0.7 for the best ®t. In this orientation, the four RC light subunit helices L A , L B , L C and L E are superimposed on four relatively dense regions separated by ~10 A Ê in the RC map (marked in red in Figure 6A). The long axis of the RC model coincides approximately with the major axis of the elliptical LH1 ring. The pseudo 2-fold axis of the RC running through the Fe and between the `special pair' of Bchls lies within 2±3 A Ê of the crystallographic 2-fold axis. With the uncertainties that arise from calculating model density at low resolution in such a complex environment of lipid, protein, pigment and glucose, the uncertainty in orientation must be at least T 3 ° . However, within this degree of uncertainty, the planes of the Bchl porphyrin rings make an angle of ~43 ° with the major axis of the LH1 ring. There are few data on the detailed nature of the interactions of the RC with the LH1, although cross- linking experiments suggest that L, M and H subunits all interact in some way with LH1 subunits (Peters et al ., 1983). In our model, within the membrane, the only RC transmembrane helices that could interact with the putative LH1 a helices would be L A , M A and possibly L B and M B ; all other helices are much further than 10 A Ê from their nearest LH1 neighbours. A near-straight line can be drawn linking the centres of gravity of two diametrically opposed LH1 a subunits and the L and M helices. This would be consistent with a helix±helix packing arrangement of L A and M A against nearest-neighbour LH1 a subunits. This study represents the highest resolution view, to date, of any RC±LH1 complex from a purple bacterium. The previous study by Karrasch et al . (1995), which indicated that R.rubrum forms a closed LH1 ring, raised the question of how quinone transfer takes place between the apparently fully encircled RC and the spatially distant cytochrome bc 1 complex during cyclic electron ̄ow. The projection maps of the RC±LH1 complex (Figures 3C and 5) would appear to con®rm that the RC is indeed completely enclosed by 16 identical LH1 subunits, so that the mechanism of quinone transfer remains unclear. This is in the context of work on the PufX polypeptide from R.sphaeroides and Rhodobacter capsulatus , which has shown that it facilitates the transport of reducing equivalents from the RC to the bc 1 complex (Farchaus et al ., 1990, 1992; Lilburn et al ., 1992). It has been shown that PufX is unnecessary in strains that cannot assemble a normal LH1 complex (McGlynn et al ., 1994, 1996). It has been suggested that PufX might affect the structure of the LH1 so as to allow the free diffusion of quinone, perhaps even substituting for one or more LH1 subunits (Bopp et al ., 1999). Moreover, Jungas et al . (1999) have proposed that the functional photosynthetic unit in R.sphaeroides is made up of a dimeric RC±LH1±PufX assembly with the two RCs only partially encircled by LH1 subunits, this open assembly presumably allowing the free movement of quinone from RC to bc 1 complex. However, the resolution was not suf®cient to reveal individual LH1 subunits. To date, no such assembly has been seen in R.rubrum and no equivalent of the pufX gene has been found. It has been proposed that the functional RC±LH1 complex might incorporate an additional small (4 kDa) polypeptide component, W (Ghosh et al ., 1994), and that this might form a quinone channel (Walz and Ghosh, 1997; Stahlberg et al ., 1998). However, no amino acid sequence is available for this polypeptide and, within current experimental uncertainty, all 16 subunits seen in the projection of the LH1 ring (Figures 3C and 5A) appear identical. An EM analysis of the structure in three dimensions and/or a speci®c labelling experiment might help to clarify this issue. In the absence of any evidence yet for a `quinone channel' or ®xed opening in the LH1 ring, the alternative possibilities of a close approach of quinone molecules either side of the LH1 or `breathing motions' in LH1 could still be entertained, at least for quinone transfer in R.rubrum (Walz and Ghosh, 1997). Certainly, the LH1 is not a rigid assembly, as shown by the circular and elliptical forms found in this study. Fluctuations in ellipticity have also been observed in the case of LH2, and it has been suggested that there might even be a partial ...