Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum

J. Mol. Biol. (1985) 185,579 594

Dimer Ribbons in the Three-dimensional Structure of Sarcoplasmic Reticulum

Lor iana Caste l lan i 1, Peter M . D . H a r d w i c k e 2 a n d Pefcr Vibert 1

1Rosenstiel Basic Medical Sciences Research Center

and

:Department of Biology Brandeis University, Waltham, M A 02254, U.S.A.

(Received 20 November 1984, and in revised form 12 May 1985)

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca 2+- ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (~ 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by ~ 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca: +-ATPase.

1. Introduct ion

The sareoplasmic reticulum is a membrane system that generates intracellular Ca 2+ fluxes necessary for muscle contraction (for a review, see Martonosi, 1984). In response to stimulation, a rapid release of Ca 2 + from the lumen of the SR¢ raises the cytoplasmic concentration to about 10 -6 to 10-sM within a few milliseconds (Endo, 1977). During relaxation a Mg 2+-dependent Ca 2 +-ATPase protein pumps C a 2+ back into the SR against a concentration gradient (Makinose & Hasselbach, 1965). Studies of SR from rabbit skeletal muscle have shown that the Ca 2 +-ATPase, of M r ~-115,000 (LeMaire et al., 1976), represents about 80% of the total membrane protein (Hasselbach, 1974; Scales & Inesi, 1976). Fragmented SR, visual- ized by negative staining, thin sectioning or freeze- fracturing, is covered on the cytoplasmic surface by

Abbreviation used: SR, sarcoplasmic reticulum.

0022 2836/85/190579 16 $03.00/0

projections ~ 60A long and 30 to 40A wide (Ikemoto et al., 1968; Deamer & Baskin, 1969; Tillack et al., 1974; Saito et al., 1978). These projections represent part of the Ca2+-ATPase molecule, since the purified and reaggregated Ca 2 +- ATPase forms vesicles carrying projections identical to those of fragmented native SR (Hardwieke & Green, 1974; Stewart & MacLennan, 1974). An asymmetrical distribution of mass across the SR membrane has also been inferred from X-ray and neutron diffraction studies (Dupont et al., 1973; Herbette et al., 1977, 1981; Brady et al., 1981; Blasie et al., 1982). Although there is general agreement about the distribution of mass across the membrane, comparatively little is known about the arrangement of the Ca2+-ATPase protein in the plane of the membrane.

Freeze-fracture studies of rabbit SR have shown particles about 85A in diameter, believed to represent the Ca2+-ATPase, embedded in the concave (interior) fracture face (Packer et al., 1974). The ratio of three to four external surface

579 © 1985 Academic Press Inc. (London) Ltd.

580 L. Castellani, P. M. D. Hardwicke and P. Vibert

project ions per 8 5 A part icle has led to the suggest ion t h a t the A T P a s e migh t have a te t ra- meric organizat ion within the m e m b r a n e (Scales & Inesi, 1976). More recent freeze-fracture studies, however, seem to indicate t h a t the ATPase molecules are grouped into dimers in the plane of the m e m b r a n e (Napol i tano et al., 1983). Other evidence for an oligomeric grouping of the ATPase has come f rom enzymat i c and ul t racentr i fuge studies of the solubilized A T P a s e (Moiler et al., 1982), f rom cross-linking approaches (Murphy, 1976) and f rom spectroscopic invest igat ions (Hoffmann et al., 1979). More direct evidence, however, is provided by recent electron microscope studies of na t ive SR prepared f rom the s t r ia ted adduc to r muscle of the scallop, which show t h a t the ATPase is organized into r ibbons of dimers (Castellani & Hardwicke , 1983). The Ca2+-ATPase of rabbi t skeletal muscle SR can be induced to cluster into similar dimeric a r rays by t r e a t m e n t with v a n a d a t e (Dux & Martonosi , 1983a; Buhle et al., 1984; Taylor et al., 1984a).

We have previously repor ted (Castellani & Hardwicke, 1983) t h a t negat ive ly s tained prepara- t ions of f r agmen ted scallop SR show a va r i e ty of crystal l ine forms, including tubules closely related to those observed in thin sections of whole scallop muscle. We describe here the three-dimensional s t ruc ture of SR membranes ob ta ined by compu ted helical recons t ruc t ion methods f rom tubules filled with stain. We also compare this three-dimensional s t ructure with improved two-dimensional maps calculated f rom electron microscopic images of f lat tened SR tubules recorded using low-irradiat ion procedures. These results indicate t h a t the tubules are composed of d imer r ibbons of Ca2+-ATPase molecules running diagonal ly a round the tube wall.

2. Materials and Methods

(a) Preparation of sarcoplasmic reticulum vesicles

Live sea scallops (Placopecten magellanicus) were obtained from the Marine Biological Laboratory, Woods Hole, MA. SR vesicles were isolated from saponin-treated striated adductor muscle as described by Castellani & Hardwicke (1983). Since this procedure involves the use of 20 mM-sodium phosphate, and reports have indicated a possible effect of phosphate in inducing ordered arrays on the surface of rabbit SR vesicles (Dux & Martonosi, 1983b), an alternative preparative method using a buffered solution containing l0 mM-TES, 100 mM-NaC1. 8 mM-MgC12, l0 mM-EGTA, with or without ATP was also used.

(b) Electron microscopy

To prepare sections of whole muscle, strips of detergent-skinned scallop adductor muscle kept under tension were fixed with glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated in a series of acetone/water mixtures, and impregnated with Araldite as described by Bennett & Elliott (1981). Thin sections, placed onto grids, were double stained with 1% lead

acetate for 10 min and 2°/o uranyl acetate for 20 min at room temperature.

For negative staining of isolated SR, one drop of freshly prepared SR suspension (0.2 to 0"5 mg/ml) was placed for 20 s onto a 400-mesh copper grid coated with a thin carbon film. The grid was washed with several drops of relaxing medium followed by 100mM-ammonium acetate. The preparation was stained with 3 drops of aqueous 2% uranyl acetate. Excess stain was drained with filter paper. This procedure was performed with similar results both at room temperature and at 4°C.

Electron micrographs of sections of whole muscle and of stain-filled SR tubes were recorded in a Philips 301 electron microscope, fitted with an anti-contamination device and calibrated using tropomyosin Mg 2+ para- crystals. A minimal dose procedure, as described by Baker & Amos (1978), was used to record electron micrographs of flattened SR tubes. All micrographs were recorded at a nominal magnification of 45.000 x.

(c) Image analysis for three-dimensional reconstruction

Micrographs of stain-filled tubes were grouped accord- ing to tube diameter, and optical diffraction patterns were recorded on film in a surveying optical diffractometer (Salmon & DeRosier, 1981). Images giving the clearest diffraction patterns (Baker & Amos, 1978) were selected and digitized on a 512 × 512 pixel array using an Optronics P1000 photoscan rotating drum microdensito- meter (Optronies International, Chelmsford, MA) with a raster (25#m step) corresponding to ~ 5.75A in the image. Suitable areas, displayed on a 256 gray level television monitor (Grinnell Systems Corp., Santa Clara, CA), were boxed using the television screen cursors and the floated density arrays were Fourier transformed on a VAX 11/780.

The helical symmetry was determined for tubes of different diameters by fitting a 2-dimensional reciprocal lattice to the transforms of the near and far sides of the tube. Images in which both sides contributed to the same reflection were rejected. Tubes of 2 helical symmetries were selected for averaging layer-line data. For each of the 2 helical symmetries, 4 tubes were used to determine the layer-line amplitudes and phases. Location of the best phase origin and the amount of tilt out of the plane of the micrograph were determined for each image. The amplitude-weighted phase residual relating the peaks corresponding to the near and far sides of the tube, calculated for 7 layer-lines, was low for each set of tubes (see Table 1). Only the data sets corresponding to the far side of the tube were used for layer-line averaging since they consistently showed a better preservation due to the combined support provided by the carbon film and the stain. (The hand of the surface lattice is known from examination of shadowed tubules (Castellani & Hardwicke, 1983).) The average layer-line data were calculated by vector addition after appropriate rotation and translation to bring the phase origins of the individual data sets into correspondence. The amplitude- weighted root-mean-square phase difference between individual data sets and their average was ~ 50 ° (Table 1) (Amos & Klug, 1975). A significant polarity is reflected in the R values (up to 85 °) obtained when a tube with inverted polarity is compared with the calculated average, although comparison of individual data sets with that of a reference particle showed only a small polarity. No significant improvement of the R values was obtained by further averaging. Layer-line ampli-

3-D Structure of Sarcoplasmic Reticulum 581

Table 1 Parameters for evaluating individual data

relative to the average

Tubule ~o Qo Rsc R ° R°~ $

44594 -- 1 "0 4 0'92 53-8 81 "9 44587 + 1-0 6 1'07 48-2 85"7 49685A - 1-5 5 1.06 58.1 78.0 49685 + 2-0 7 1.07 55-1 84.2

49677 0-0 4 1-0 57-0 80.2 46630 + 1-0 9 1.05 55.7 87.1 46683 --0.5 23 1-0 56.8 84.2 46686 -- 1.0 14 1.0 50-2 86-7

gt °, Apparent filament tilt that minimizes Q; QO, normalized amplitude-weighted mean phase residual between peaks on either side of the meridian (equator not included) (Dcl%sier & Moore, 1970): Rsc, radial scaling factor relative to average; R °, normalized amplitude-weighted root-mean-square phase difference between individual sets of layer-lines and their average (Amos & Klug, 1975); RT$, residual when an individual data set is compared with the average after polarity reversal.

tudes and phases of the average da ta sets were edited to remove small peaks with rapidly f luctuat ing phases at both large and small reciprocal radii along the layer-lines. The highest resolution da ta included were at 0"035 A -

radially and 0-029A-1 axially. The da ta indicated in Fig. 5 were used for calculating the 3-dimensional maps. The equatorial da ta were included with 0-85 and 0"65 weight, respectively, for the 2 helical symmetries.

(d) Image analysis for 2-dimensional averaging

Micrographs of f lat tened tubes were screened with a surveying optical diffractometer and only those giving 2 dist inct sets of reflections from the 2 sides of the tubes were used for analysis (Castellani & Hardwicke, 1983). Selected areas were digitized and Four ier t ransformed as described above. Transform ampli tudes were displayed on the television screen, and best co-ordinates of the diffraction maxima for a near-orthogonal reciprocal latt ice were determined by a least-squares fit of the peak positions for ~ l0 reflections. A single ampli tude, in tegrated and corrected for background, and the corresponding phase measurement at each peak position, were calculated using the method described by Baker & Amos (1978). This Four ier averaging of up to 60 uni t cells generates s t ructure factors to a resolution l imit of

25 A. Sets of s t ructure factors from near and far sides of 3 different images were put on a common scale and summed to calculate the averaged reconstructed image as described by Baker et al. (1983).

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Figure 1. Elect ron micrographs of th in (a) longitudinal and (b) t ransverse sections of embedded scallop s t r ia ted adductor muscle. (a) The SR membrane system appears as a complex network of tubules and cisternae. Magnification: 40,000 ×. (b) SR tubules show nearly circular cross-sections and different diameters. Magnification: 40,000 ×.

582 L. Castellani, P. M. D. Hardwiclce and P. Vibert

3. Results

(a) Electron microscopy

The SR membrane system in scallop striated muscle appears to constitute a complex network of tubules and cisternae, as shown by thin sections of the whole muscle (Fig. l(a)). In cross-section, the tubules show nearly circular profiles and variable diameters (Fig. l(b)). Negatively stained preparations of fragmented SR show a variety of forms, including tubules, indicating tha t the gentle homogenization and purification procedure causes only a limited disruption of the SR system. Both tubules and vesicles show a regular surface array of ribbons composed of dimeric morphological units. The superposition of the front and back halves of

the particle produces an apparent diamond-shaped array. Substitution of sodium phosphate by TES during the purification procedure does not alter the crystalline appearance of the preparation, indicating that phosphate does not have a direct effect on the surface order of these scallop membranes, as also reported by Ferguson et al. (1985).

In a field of negatively stained fragmented SR, both tubules filled with stain (Fig. 2(a)), and tubules flattened onto the grid (Fig. 2(b)) are observed. Stain-filled tubules show a bright stain- excluding edge due to the edge-on view of the membrane wall and a darker middle region reflecting the large amount of stain accumulated on the inside. The diameters of the tubules range from 400 A to 800 A; this variation is par t ly accounted

Figure 2. Images of negatively stained scallop SR. (a) Tubules filled with stain showing the bright stain-excluding areas at the edges. (b) Tubules flattened onto the grid are uniformly stained. (c) Vesicles showing the ribbon arrangement of the morphological units. Arrowheads indicate the pairing of projections originating from the same ribbon.


for by limited flattening of the tubules, but also reflects the intrinsic difference in tubule sizes observed in transverse sections of whole muscle (Fig. l(b)). Some of these tubules show both stain- filled and flattened segments (see, e.g. Fig. 2(b), last image)• The difference in diameter between the two segments of the same tube clearly indicates tha t the stain-filled portion is approximated by a cylinder. A fringe of projections can be easily seen at the edges

of the stain-filled tubules, although the individual projections are not as clearly visible as in spherical vesicles (Fig. 2(c)). Occasionally, projections originating from the same ribbon on the surface of the vesicles appear to be joined together in pairs. Flat tened tubules appear uniformly stained and are consistently of large diameter (Fig. 2(b)). Their edges, although not smooth, do not show projections as clearly as do the stain-filled tubules.

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(b) Stain-filled tubules

(i) Indexing of the diffraction patterns

Examples of images and their computed Fourier transforms for the two classes of stain-filled tubules used for three-dimensional reconstruction are shown in Figure 3. The pattern of reflections, which on average extends to a resolution of 30 A radially and 35 A axially, shows a marked system of layer-lines indicative of a well-defined helical symmetry. Furthermore, the amplitude of the transform along the layer-lines consists of a principal maximum with a series of subsidiary peaks only on the side away from the meridian (see Fig. 3(b) and (e), and Fig. 5), indicative of a transform from a tubular object (Waser, 1955). To

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determine the helical parameters of the two classes of tubules, we indexed each pa t te rn of reflections in terms of two reciprocal space lattices (related by a mirror line along the meridian) tha t originate from the front and back halves of the tubule (Moody, 1967; Kiselev & Klug, 1969; Baker & Caspar, 1984). (We call the back half the side of the particle in contact with the carbon support film.) The first meridional reflection, which occurs at the point along the meridian where the two lattices overlap, defines the indices of the circumferential vector ([7) in the surface lattice of the tube and the corresponding axial repeat vector (Co) (Fig. 4) (Baker & Caspar, 1984). The circumferential vectors are V = 5 ~ - 6 b and V = 65-5b, respectively, for the two classes of tubules analyzed (Fig. 4(a) and

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Figure 4. Surface lattices for the 2 classes of tubules of (a) 53/10 and (b) 63/11 helical symmetry; (c) and (d), their corresponding (n,/) plots describing the diffraction from these lattices. The surface lattices are drawn, by convention, as though seen from inside the tubule: the major stain~lled grooves that run along the (5,3) or (6,3) helical tracks therefore appear left-handed. The circumferential vector V and the axial vector Co are, respectively, the 2 axes of the surface lattices in (a) and (b) (see Baker & Caspar, 1984). The unit cell vectors ~ and b are indicated by arrows. Various helical tracks are labeled with the (n, l) values. The first meridional reflection corresponding in (c) to 6~.* + 5b* (for the 53/10 type) and in (d) to 55"+6b* (for the 63/11 type) are indicated. The filled circles indicate the reflections used for obtaining the averaged layer-line data.


(b)). These vectors correspond to 5-3 and 5-7 units in one turn of the genetic helix, reflecting the observed difference in diameters between the two classes. (Note tha t the "uni t" defined here corresponds to the unit cell of the surface lattice, which contains two morphological units.) The indices of the circumferential vector, however, may not be uniquely defined when the circumference of a tubular structure is large compared to the unit cell dimensions (Baker & Caspar, 1984). Alternative choices of the circumferential vector were tested, but they did not give a good fit to the tubule diameters measured from the original micrographs or to the phase of certain reflections in the Fourier transform. Some variability is observed in the ratios of layer-line axial spacings, indicating that random distortions may have been intro- duced by shrinkage in the process of negative staining and during the recording of the images. The ratios of the average layer-line spacings to the axial spacing of the near-meridional reflection (Table 2), however, are in good agreement with the defined selection rules, 1 = 5 3 m - 10n and l = 63m + 1 ln, for the two classes of tubules. The two structures can therefore be described in terms of 53 units in l0 turns of a left-handed genetic helix, and 63 units in 11 turns of a right-handed genetic helix. This description can be more readily understood by looking at the two-dimensional net (Fig. 4(a) and (b)) obtained from the reciprocal lattice (Fig. 4(c) and (d)). (The back half of each tubule is used to derive the two-dimensional net since this half of the particle is usually better preserved.) There is some variation in the dimensions of the unit cell of the net for the two structures: 108.1(_ 8.1) × 59.2(_ 1.8) A, included angle 105.6(___4.2) ° and 112.1(_+2.5) x 62.3(_+ 1.3) A, included angle 109.1(_+3-3) ° . Note, however, tha t the fitting of a two-dimensional reciprocal lattice to a pattern of reflections originating from a cylindri- cal object can only approximate the surface lattice (Klug et al., 1958). The near-meridional reflections at axial spacings of 1/47A for the "53/10" symmetry and 1/48A for the "63/11" symmetry represent the pitch of the genetic helix used to describe each structure. The corresponding axial rise/subunit is 8.85 A and 8.4 A, respectively.

Table 2 Ratios of average layer-line spacings to axial spacing

of the near-meridional reflection

n R~,lo/R.,~ 53/10 n Rl, ll/R..t 63/11

- 16 9.29 10.00 - 17 5.42 5.5 5 3.46 3'33 6 3'66 3"67

- ] 1 2-40 2.50 - 1 1 2.22 2.20 l0 1.71 1-67 12 1.84 1.83

- 1 1 -00 1 -00 1 1 '00 1 . 0 0 4 0-78 0-77 7 0.79 0-79

Ri. 1o/R., ~ and R~, H/R.,t represent the average measured ratios of axial spacings of layer-lines identified by the Besse] order n. These values are compared with the expected values for the 53/10 and 63/11 helical symmetries applied.

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The transforms of the individual tubules of each class were averaged to improve the signal-to-noise ratio (Fig. 5). The density terms calculated from the averaged transform (Fig. 6) indicate tha t the stain- excluding region is radially distributed between 65 and 203 A for the 53/10 class, and between 70 and 216 A for the 63/11 class. The maximum radii for the two mass distributions are in good agreement with the values of 195 to 207 A and 218 to 230 A measured directly from the images of the particles used.


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Figure 6. Some of the averaged individual density terms [g,, l[ identified by their (n, l) indices for the (a) 53/10 and (b) 63/11 helical symmetry tubules. The n = 0 term suggests a maximum radius for the tubule of 203 A and 216 A, respectively (arrows). The other terms appear to be consistent with the n = 0 term for the maximum and minimum radii.

(ii) Reconstruction of tubule with 53/10 helical symmetry

The solid model of the three-dimensional reconstruction calculated from the average transform is shown in Figure 7. The tubule consists of rows of morphological units, identified as Ca 2+- ATPase molecules, coupled into five right-handed ribbons ( ~ 98 A wide), which wrap around the tube wall at an angle of ~ 42 ° to the tube axis (see also Fig. 8(c)). The ribbon appearance is conferred both by a strong bond between units along the rows (Fig. 7(a)) and by a bridge connecting the outer ends of units from adjacent rows (Fig. 7(b)). This bridge, oriented nearly parallel to the tube axis, confers a dimeric arrangement of the Ca 2 +-ATPase molecules within the ribbon (see Figs 7 and 10(a)). Deep grooves separate the ribbons (also marked as (5,3) in the surface net (Fig. 4(a)) and correspond to the pronounced right-handed striations observed in shadowed tubules (Castellani & Hardwicke, 1983).

The shape of the units is complex. In the radial direction they appear elongated and curved with a maximum chord of ~ 95A and an average thickness of 30 A (see Figs 8(c) and 9). In the axial direction they extend ~ 65 A (Figs 7 and 8(a)). The units forming the dimer, displaced axially by

34 A, are inserted in the tubule walt so that they appear to be related by a 2-fold axis normal to the plane of the membrane (see overlay in Figs 7(b),

8(a) and 10(b)). Figure 9 shows an example of two cross-sections 43 A apart, compared with the three- dimensional model and a stack of isodensity contour maps viewed down the tube axis. The units (A,B), related by the 2-fold symmetry, are connected at their outer ends by a "bridge" nearly parallel to the tube axis, and they also come into close contact at their inner ends. An additional mass of globular shape (~ 25 A in diameter) occurs at one end of the bridge, but does not appear to be par t of the basic dimer unit (Figs 8(b) and 9). This extra density breaks the 2-fold symmetry that relates the units of the dimer in projection, and appears to confer polari ty on the tubules. Different views of the solid dimer model carved out of the tubule are shown in Figure 10 with this additional globular mass removed for clarity. The volume of such dimers (2-6 x l05 A 3) corresponds closely to the expected anhydrous volume of two protein molecules with molecular weight 115,000 and partial specific volume 0"73 ml/g (2 × 1-39 x l0 s A3). The bridged units forming the dimer, given their curved shape and the contact they make at their inner ends, leave a stain-filled hole running between the bridge and the inner tubule wall (see Figs 8(c), 9 and 10(c)). Adjacent units with the same orientation form a strong contact tha t confers the ribbon appearance (Figs 7 and 8(a)).

The ribbons are held together by two different bonds that cross the major grooves in the internal par t of the tubule wall. One occurs between units with opposite orientation tha t are nearly in register axially (8.85 A displacement) (Fig. 7). The other one (conserved also in tubules with 63/11 symmetry) connects dimers displaced axially by almost their full length (Figs7 and 8(a)). There are l l connections of this type, and they appear to run through the inner ends of the dimers (Fig. 10(e)) along steep left-handed tracks (marked as the ( -11 ,4 ) helices in the surface net in Fig. 4(a)).

The luminal surface of the membrane wall appears quite smooth and compact with the exception of two classes of radial stain-filled regions that connect the luminal to the cytoplasmic side of the tubule (Fig. 7 and Fig. 9(a)). The major stain- filled region corresponds to the external deep grooves; and a second occurs between dimers within the ribbon. This discontinuity of the luminal surface, presumably a result of stain penetration into the lipid bilayer (Ting-Beall, 1980), does not permit the identification of a constant thickness, stain-excluding region that would correspond to the lipid bilayer. In addition, the observation that some stain-filled tubules appear to be sealed at both ends supports the view tha t uranyl acetate readily crosses the bilayer.

(iii) Reconstruction of tubules with 63/11 helical symmetry

Figure 8 shows the three-dimensional reconstruction calculated from the average transform of tubules with 63/11 helical symmetry. The overall appearance of this class of tubules is closely related


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Figure 8. Paired views at different angles of solid models of the averaged helical reconstructions for the 2 classes of tubules; 53/10 type ((a) and (c)) and 63/11 type ((b) and (d)). One section of the solid model corresponds to 4.3 A. The overall shapes of 2 bridged units constituting the dimer are outlined in (a) and (b); 5 and 6 deep right-handed grooves, respectively, separate the dimer ribbons wrapping around the tubule wall (asterisks in (c) and (d)). The additional globular mass attached at one end of the bridge is outlined in (c) and (d) by broken lines.

to tha t of the smaller diameter tubules (53/10 type). Their larger diameter is reflected in the increased number of units in one turn of the genetic helix (5.7 dimers/turn instead of 5-3). Note tha t here the genetic helix, which has no necessary bonding significance, is of opposite hand to tha t in 53/10 tubules (see also Fig. 4). The ribbons of dimers, now six in number (Fig. 8(d); see also Fig. 4(b)) are easily identifiable~ and again run in the tubule wall at an angle of ~ 40 ° to the axis. The units forming the dimers have a similar overall shape, volume and orientation to those in the 53/10 structure, although

they appear to be slightly longer axially (73 A, rather than 65A) and are displaced by ~ 35A (Figs 8(b) and 10(b) and (d)). Ribbons of dimers (~ 104 A wide) are again formed by bonds between units with the same orientation and by vertical bridges in the outer par t of the wall (Figs 8(b) and 10(b)). The additional globular mass is at tached at one end of the bridge (Fig. 8(d)), as observed in 53/10 tubules, indicating that it may be a true feature of SR tubules. The bridge in this class of tubules seems to run at a slightly different angle, so that its central region appears weaker (Fig. 10(b)).


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Unlike the situation in the smaller tubules, only one type of bond appears to hold the ribbons of dimers together. This bond~ which appears to be conserved in the two structures, occurs between axially displaced dimers and follows steep left- handed helical t racks (Figs 8(b) and 10(d); and the - l l , 5 t racks in Fig. 4(b)). The internal wall shows features similar to those described for tubules of smaller diameter (Figs 8(d) and 10(d)) and poses a similar problem in the identification of the lipid bilayer (see above).

(c) Flattened tubules

A typical example of a negatively stained flattened SR tubule, recorded by low-irradiation electron microscopy, and its computed diffraction pa t t e rn are shown in Figure 11. The profile of the diffraction spots, consisting of a central m a x i m u m with a series of subsidiary peaks at both tails, suggests tha t the diffracting object can well be approximated by a two-dimensional crystal (Amos et al., 1982). The pa t te rn of reflections extends to a resolution of ~ 25 A, and can be indexed by two lattices related by a vertical mirror line (Castellani & Hardwicke, 1983). The two lattices are produced by the superimposed front and back half of the tubules. Only tubules tha t give non-overlapping diffraction pa t te rns from the two halves of the particle have been used for two-dimensional reconstruction. The search for the phase origin in each da ta set analyzed gave four equivalent min ima at (0,0), (a/2,0) (0,b/2) and (a/2,b/2) with residuals ranging between 5.7 ° and 9.9 ° . These results strongly indicate tha t these arrays contain four 2-fold rotat ion axes normal to the plane of the membrane . Such a 2-fold symmet ry , however, was not enforced in calculating the reconstructed maps. The average reconstructed image, shown in Figure l l (c) , represents the sum of six sets of s t ructure factors from three tubules (front and back for each tubule). The R factors between individual da ta sets and their sum ranged between 0-182 and 0.263. The unit cell, oriented with the short axis a t an angle of

58 ° to the tubule axis, has average dimensions of (130.47( _ 4.3) × 62-33( +_ 1-4)) A, included angle (93.44_+0-71) ° and contains two morphological units of about ~ 45 × 70 A tha t will be referred to

Figure 9. View down the axis of the 53/10 type tubule: (a) solid model; (b) stack of isodensity contour maps (cross-sections cut at 8"6 A intervals) showing 2 units forming the dimer identified as A and B and the density distribution across the membrane wall; and (c) 2 cross- sections 43 A apart showing nearly equivalent views of the 2 units forming the dimer. The region where the external bridge is formed is arrowed and the internal contact between the units can also be seen.

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Figure 10. (a) and (b) Cytoplasmic; (c) and (d) oblique view (cf. Fig. 7(b)) ~ 30 ° to axis of tube; and (e) and (f) luminal views of a dimer carved out of 3-dimensional reconstructions of tubules with 53/10 (left) and 63/11 (right) helical symmetries. The additional mass present at one end of the bridge has been omitted for clarity. One section of the model corresponds to 8"6 A. A and B are the monomers related by 2-fold symmetry within a ribbon. In (a) and (b) the bridge linking the 2 units is outlined and the arrowheads indicate the direction of the conserved bonds between units along the ribbon directions. Outlines in (c) and (d) show the orientation of the A and B-type monomers; the stain-filled region between the monomers can also be seen behind the external bridge; (e) and (f) show the 2 units (A, B) also coming into close contact in the internal region of the membrane wall. Note that here the dimer is viewed from the inside of the tube.


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Figure l l . (a) Low-dose electron micrograph of flattened SR tubule; (b) its computed diffraction pattern; (c) the average reconstructed image calculated from 3 tubules; and (d) part of the dimer ribbon surface envelope calculated by helical reconstruction from 53/10 type tubules. The pattern of reflections shown in (b), originating from front and back sides of the tubule, extends to a resolution of ~ 25 A. The average reconstructed image shown in (c) shows dimer ribbons running at an angle of ~ 58 ° to the tubule axis. The unit cell of average dimensions (130 A × 62 A) included angle of 93 ° (shown by outline). Asterisks show the direction of the bond between dimers from adjacent ribbons. Note the similarity in the shape of the units and in the bond along the ribbon between the (c) 2- and the (d) 3-dimensional reconstruction.

as a dimer. The map is characterized by ribbons of dimers running diagonally across the tubule, staggered by about half a unit and separated by grooves filled with stain (Castellani & Hardwicke, 1983). The shape of the units, however, is more elongated than previously shown and strongly resembles the map obtained from rabbit SR treated with vanadate (Taylor et al., 1984a). These results indicate that the difference previously observed between scallop and rabbit SR was due to a

combined effect of radiation damage and limited resolution. The difference in the unit cell dimensions between maps obtained from images recorded with low-dose and conventional microscopy (Castellani & Hardwick@, 1983) may also be due to shrinkage induced by radiation.

The present two-dimensional map is consistent with the three-dimensional model obtained from helical reconstruction of stain-filled tubules. The strong contact between units with the same


orientation gives rise to the ribbon appearance also observed in three dimensions (see Fig. 1 l(c) and (d)). The non-uniformity of the stain in the grooves between ribbons suggests the presence of a weak contact between dimers that follows steep left- handed tracks, strongly resembling the inter-direct bond described in the three-dimensional maps (Fig. ll(c) and (a)). Neither the bridge between the units of the dimer within a ribbon nor the globular mass at one end of the bridge is clearly visible, however, probably because of the high contrast that results from projecting the structure in two dimensions. Nonetheless, the narrow lines of stain within the ribbon are not uniformly distributed, suggesting a possible connection between the units of the dimer. Furthermore, the small pool of stain observed between these units is probably due to stain accumulation in the hole beneath the outer bridge described in the three-dimensional maps.

4. Discussion

SR isolated from scallop striated muscle reveals an ordered surface array of morphological units, grouped into dimers, which have been interpreted as Ca2+-ATPase molecules. The tubular forms observed in these preparations strongly resemble the tubular system observed in thin sections of the whole muscle indicating that tubules are not an artifact of the preparation procedure. Substitution of phosphate with TES in the preparation of these membranes does not affect their crystalline surface appearance, as also reported by Ferguson et al. (1985) for skeletal SR. Freeze-dried preparations of scallop SR, shadow-cast with platinum, show features similar to those observed in negatively stained images (Fcrguson et al., 1985). Grooves running diagonally around the wall of the SR tubular system have also been observed in freeze- fracture deep-etched preparations of the whole scallop muscle (C. Franzini-Armstrong, personal communication) and in thin sections of muscles rapidly frozen and freeze-substituted (R. Craig, personal communication). These results, taken together, indicate that the ordered surface array of isolated scallop SR is not an artifact induced by the preparative procedure.

Three-dimensional reconstructions calculated from stain-filled SR tubules confirm the results obtained from two-dimensional analysis of tubules flattened onto the grid (Castellani & Hardwicke, 1983; Taylor et al., 1984a; Bnhle et al., 1984). Five or six deep right-handed grooves run along the tubule wall for the two classes of particles analyzed. This difference in the number of grooves reflects the different diameters of the particles, but the spacing between the grooves remains quite constant, suggesting that the tubules are built on related principles. Ribbons of dimers occur between the major grooves. The units forming the dimer, interpreted as individual ATPase molecules, are displaced axially in the membrane wall and appear to be related by a 2-fold axis normal to the plane of

the membrane. This description is consistent with the two-dimensional map obtained from flattened tubules in which there is a clear indication of a 2- fold axis relating the morphological units of the dimer. This 2-fold symmetry was not, however, enforced in the reconstructed three-dimensional structure to avoid possible obliteration of fine details. A prominent bridge joins the two units in the outer part of the membrane wall, oriented nearly parallel to the tubule axis. An indication of bridging between the Ca2+-ATPase molecules is also seen in freeze-dried, rotary-shadowed SR preparations (Ferguson et al., 1985) and in negatively stained vesicles where the projections at the edges appear to be joined together. Similar bridging between the two morphological units has been described in three-dimensional reconstruction calculated from tilt series of rabbit SR (Taylor et al., 1984b).

The overall shape, dimensions and volume of the Ca2+-ATPase molecules from the two reconstructions appear quite similar, although the angle at which the units are inserted in the membrane wall seems to vary. The units in the 63/11 tubules (larger diameter) appear to be oriented nearly perpendicular to the membrane plane, whereas the ones in the 53/10 tubules (smaller diameter) lie at an angle that brings the outer regions of the molecules closer together. This difference in orientation may account for the difference in shape of the bridge joining the dimer, which appears bulkier in the tubules with 53/10 symmetry. The bridging contact thus appears to be flexible. Additional globular mass is observed at one end of the bridge in both reconstructions suggesting that the 2-fold symmetry observed in flattened tubules is not preserved in three dimensions. This density cannot easily be accounted for as part of the Ca 2 +-ATPase molecule since it is present in a ratio of one to two Ca2+-ATPase molecules. Its asymmetrical distribution, however, may account for the polarity of the tubules apparent in the three-dimensional transforms. Although the main constituent of SR membranes is the Ca2+-ATPase, other proteins are present. I t seems reasonable to assume that one or more of these additional proteins may account for the extra mass observed in our three-dimensional reconstructions.

The single Ca 2 +-ATPase molecule has an average thickness of ~ 30 A, a maximum chord of 95 A in the radial direction and a length of ~ 65 A in the plane of the membrane. Its projection in the membrane plane strongly resembles the overall triangular shape of the units seen in two- dimensional maps calculated from flattened tubules by low irradiation methods (Fig. 11 (c); Taylor et al., 1984a). Its length in the radial direction is consistent with the measurements obtained from X- ray and neutron diffraction studies (Dupont st al., 1973; Blasie et al., 1982). The fringe of projections observed in negatively stained SR vesicles from both scallop and rabbit preparations, therefore, represents the portion of the Ca 2 +-ATPase molecule

3-D Structure of Sarcoplasmic Ret iculum 593

projecting out of the membrane wall. However, we do not have direct information on the localization of the lipid bilayer with respect to the ATPase molecules. The tubules used for helical reconstruction are stained on both the inside and outside surfaces. The bilayer might therefore be expected to appear as a continuous stain-excluding region, but such a region is not visible in the three-dimensional reconstruction. The difficulty in obtaining a clear identification of the lipid bilayer may be the result of several factors. Stain penetrat ion through the membrane m a y be responsible for disrupting the ar rangement of the bilayer. Fur thermore , the high contrast produced by the stain accumulated in the deep grooves and in the interior of the s tructure m a y also contribute to obscuring such a signal. A possible assignment for the localization of the lipid bilayer can, however, be suggested from the length of the projections in negat ively stained preparations ( ~ 70 A), the thickness of the SR membrane wall from thin sections (Saito el al., 1978), and the weak peak at low radius present in the mass distribution te rm from the equator of the two classes of tubules. The lipid bilayer m a y therefore be placed in the inner par t of the tubule wall (luminal side), so tha t the Ca2+-ATPase molecules are inserted into but do not protrude through the luminal leaflet of the bilayer. This localization would account for the smooth surface of the internal membrane wall in the three-dimensional reconstruction, and it is consistent with the freeze- fracture studies of isolated and in situ SR (Packer et al., 1974; Franzini-Armstrong, 1975), and with X- ray and neutron diffraction measurements {Blasie et al., 1982; Brady etal. , 1982).

Comparison of the ar rangement of the Ca 2+- ATPase dimers in tubules with different diameters and helical symmetr ies reveals conserved and variable contacts between the units, which may be responsible for the format ion of assemblies of varying size. The strong bond (A-A or B-B) tha t gives the ribbon appearance to the tubules (and also to spherical vesicles) is highly conserved. This bond is also conserved in flattened tubules tha t show the typical "double r ibbon" appearance. Of the two bonds tha t hold together adjacent ribbons in tubules with 53/10 helical symmet ry , only the one following steep left-handed helical t racks and running through the dimers is conserved also in the 63/11 structure; moreover, it does not appear to be disrupted by flattening of the tubules (Fig. ll('c)). This bond m a y be responsible for the formation of tubules of different diameters, and possibly accounts for the flexibility required for the SR tubular system to follow changes of sarcomere length during contraction.

The SR membrane system ensures not only the fast removal of Ca 2+ from the cytosol, which induces muscle relaxation, but also the rapid passive release of calcium ions necessary for muscle activation. The results presented here indicate tha t the membrane is densely packed with protein, suggesting tha t both active up take and passive

release of Ca 2 + m a y occur via protein channels. The stain-filled regions transversing the membrane wall in correspondence with the deep grooves are probably due to stain penetrat ion and disruption of the lipid region. The interactions between the Ca 2 +- ATPase molecules, such as the bridging within each dimer on the cytoplasmic surface, provide a physical basis for the possible functional role of the association of the Ca 2 +-ATPase into oligomers.

We thank Dr Timothy Baker for helpful advice, Drs Carolyn Cohen and Andrew G. Szent-GySrgyi for valuable suggestions and the use of facilities, Drs David DeRosier and Donald L. D. Caspar for comments on the manuscripL Judith Black for the photography and Louise Seidel and Beth Finkelstein for the typing. This work was supported by grants from NIH (AM17346 to C.C.; AM15963 to A.S.-G.), NSF (PCM 824)2516 to C.C. and P.V.), and MDA (to C.C. and A.S.-G.). L. Castellani was supported by a Fellowship from the Charles A. King Trust of New England. Funds to purchase and maintain the VAX 11/780 computer were obtained from a Shared Instrumentation Grant 3-R01-GM21189-09S1 awarded to D. J. DeRosier by the National Institutes of Health.

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Edited by H. E. Huxley

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Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum