Structural changes in thin filaments of crab striated muscle

.I. Mol. Bid.

(1979) 127, 191-201

Structural Changes in Thin Filaments of Crab Striated Muscle Y.

MA&DA?,

I. &IATSUBILRA

AND

N.

YAW

Department of Pharmacology Tohoku University School of Medicine Seiryo-machi, Sendai, Japan 980 (Received 1 March

1978)

J,ow angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits iii the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between 26/12 and 28/13. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to 28/13. In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 x 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity dist,ributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments. The muscle in rigor gave meridional and near-meridional reflections at orders of 2 x 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.

1. Introduction A regular arrangement of troponin in the thin filaments of muscle was first demonstrated by Ohtsuki et al. (1967) with electron micrographs of myofllaments treated with anti-TN$ antibodies. This finding led Ebashi et al. (1969) to propose a thin filament model in which TN molecules are located at about 40 nm intervals on each of the two tropomyosin strands lying in the grooves of the long-pitch actin helices. This was later confirmed by Rome et al. (1973) who showed, using glycerinated vertebrate muscle, that a meridional X-ray reflection at 38.5 nm was enhanced by treating the muscle with anti-TN antibodies. Recently, Ebashi (1972) and Ohtsuki (1974) have proposed a more detailed model in which the pair of TN molecules (on different TM strands) found at 38.5 nm intervals along the thin filament are not aligned transversely, but are displaced axially by 2.73 nm relative to each other. This, however, has not yet been confirmed experimentally. On activation of muscle, TM strands move towards the centre of the helical grooves (Haselgrove, 1972: Huxley, 1972; Parry & Squire. 1973). The same structural change t Present

Nagoya,

address:

Institute

of Molecular

Biology,

Faculty

of

Science,

Nagoya

University,

Japan.

1: Abbreviations

used:

TN,

troponin;

TM,

tropomyosin. 191

002%2836/79/02191-11

$02.00/O

0 1979 Academic

Press Inc. (London)

LM.

Y. MAEDA,

192

I.

MATSUBARA

AND

N. YAGI

has been shown to occur in paracrystals of reconstituted thin filaments (Gillis & O’Brien, 1975; Wakabayashi et al., 1975). An additional structural change takes place in these paracrystals; the symmetry of the actin helix changes when the Ca2+ concentration is altered (Gillis & O’Brien, 1975). The same change might be expected to occur in muscles (O’Brien et d., 1975), but there is no firm evidence for this. The present experiments have been carried out to investigate the detailed structure of the thin filament in a muscle which gives abundant X-ray reflections ascribable to thin filaments. As a result, we have obtained evidence supporting Ohtsuki’s detailed model of the arrangement of TN molecules in the thin filaments, and have detected in the muscle the change in symmetry of the actin helix. The muscle used was from crab leg, in which the thin filaments are twice as long as those of vertebrate skeletal muscle, and in which there are 12 thin filaments around each t,hick filament in the overlap region (Ma&la, manuscript in preparation). Results of gel electrophoresis in the presence of sodium dodecyl sulphate have shown that the muscle contains TN, TM and paramyosin (Ma&la, unpublished results).

2. Methods (a) Preparation

of

glycerol-extracted

muscles

A flexor muscle (0.6 to 1 mm thick), together with the shell and the apodeme, was dissected from a walking leg of the crab Plagusia dentipes. Using the diffraction of a laser light (X = 0.6328 pm), the sarcomere length of the muscle was adjusted to 5.0 to 5.5 pm, at which 75 to 85% of the length of each thick filament overlaps with the thin filaments. The muscle was held at that length, and was kept in a glycerol solution (50% glycerol; 100 m&r-KC1 ; 1 mM-MgCl, ; 10 m&r-imidazole chloride: pH 7.2) at 0 to 4°C for one day. Then the muscle was transferred to a specimen chamber filled with a salt solution (100 mMchloride; pH 7.2). The specimen chamber was a KCI; 1 mM-MgCl, ; 10 mM-imidazole Perspex box with inside dimensions 7.0 cm x 2.5 cm x 0.8 cm, and had thin mylar windows to allow passage of X-rays through the muscle. The temperature of the salt solution was kept at 4’C with a thermoelectric cooling module (De La Rue Frigistor, type 12-15G) attached to a copper block surrounding the chamber. Some glycerol-extracted muscles were treated with the myosin extracting solution (Hanson & Huxley, 1957) for 2 h, before being transferred to the specimen chamber. (b) Preparation

of living

resting

muscles

A fresh flexor muscle, isolated in the same way, was mounted in the specimen chamber at a sarcomere length of 5.0 to 5.5 pm. The chamber was filled with the normal crab Ringer solution (Fatt & Katz, 1953), which was kept at 4°C and continuously oxygenated.

(c) X-ray diffraction A mirror-monochromator focusing camera of the type described by Huxley & Brown (1967) was used. The X-ray source was a fine-focusing rotating-anode generator (Rigaku RU200) operated at 40 kV with a tube current of 35 mA (nominal focal size 1 mm x O-1 mm, viewed at an angle of 6”). The diffraction pattern was recorded on X-ray film (Sakura N) with a specimen-to-film distance of 35 cm. The exposure time was 5 h for recording the equatorial pattern, and 24 h for the axial pattern.

3.

(a)

(i) Equatorial Eight

2,0; 2,l;

Results

Living

resting muscle

rejections

reflections

were

3,0; 2,2+3,1;

observed

on the equator,

4,0 and 3,2+4,1

and were

reflections

from

indexed

as the

the hexagonal

1 ,O; 1,l;

filament

STRUCTURAL

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193

lattice. At the sarcomere length of 5.0 to 5.5 pm, the spacing between the 1,0 lattice planes was 51.3 to 51.9 nm, and the intensity of the I,0 reflection was stronger than that of the 1.1 reflection. (ii) Hejlections

assigned to the thick j&men.%

\:ery strong reflections, which could be indexed as the lst, 2nd, 3rd and 4th orders of the 14.4 nm myosin periodicity, were observed on the meridian (Fig. I(a) and (b)). The 14.4 nm reflection was accompanied by strong off-meridional reflections at the same axial spacing, forming a layer-line. No other myosin layer-lines were observed SO t,hat we were not able to determine the helical symmetry of the thick filament. (iii)

Rfzflections

assigned

to actin

Four layer-lines which could be associated with the actin helix were seen at 36.6 nm, 5.91 nm. 5.09 nm and 2.91 nm. (The axial positions of these layer-lines were measured at their intensity peaks in the off-meridional region. With the 36.6 nm layer-line. whose intensity distribution could not be reproduced satisfactorily in Fipw l(a). t,he measurement was made at the intensity peak ranging radially from (j.05 nm - l to 0.15 nm - l.) A meridional reflection, seen at 2.71 nm, was also assigned to the actin helix. These layer-lines and t,he meridional reflection could not be indexed on 2 >*:36.6 nm, nor on 2 x 38.3 nm on which the actin layer-lines in the rigor pattern were indexed (see section (b), below). We cannot exclude the possibility that myosin might be contributing to the layerline at, 36% nm. Nevertheless, similarity of its axial breadth to those of the actin layer lines at 5.91 and 5.09 nm suggests that it arose principally, if not wholly, from actin. (iv) KeJEections assigned to TN A series of reflections indexing on 2 x 38.2 nm were observed on the meridian and in the near-meridional region up to the 9th order (Table 1). The even-order reflections (I = 2, 4, 6, 8) had small lateral spreads and showed distinct intensity maxima on the meridian. The odd-order reflections (1 = 3, 7,9), m . contrast, had large lateral spreads and did not have intensity maxima on the meridian (the radial positions of the intensity

TABLE 1 Meridional

nrd wear-vneridional

re$ectiow

ass@ed

to TN in, crab leg muscle

at rest Order of 2 x 38.2 nm 1

Measured spacing d (nm)

d x 1 (nm)

2 3 4 6 7 8 9

38.7 25.1 19.1 12.7 11.0 9.63 8.49

77.4 75.3 76.4 76.2 77.0 76.2 76.4

Values have probable

errors of approximately

lrltensity maximum on the meridian

i-i+ -

0.6%. The average value of rl x 2 is 76.4 nm.

FIQ. 1. Diffraction patterns from crab leg muscle. Mirror-monochromat.or camerawith speoimento-film distance of 35 cm. Exposure time 24 h. Scale bar 0.1 nm-’ in each photograph. (a) and (b) Patterns from living resting muscle. (a) is the second negative of (b), showing the central part of the diffraction pattern. The innermost layer-line (arrow 1) is the a&in layer-line at 36.6 nm. The actin layer-line at 6.91 run is indicated by arrow 3. The 1st.(arrow 2), 2nd- and 3rd.order reflections of the 14.4 nm myosin periodicity are clearly seen. A series of meridional and near-meridional reflections at orders of 2 x 38.2 nm can be seen up to the 9th order (8.5 nm). (c) and (d) Patterns from glycerinated muscle. (c) is the second negative of (d). The innermost layer-line (arrow 1) is the actin layer-line at 38.3 nm. Arrows 2 and 3 indicate the myosin reflection at 14.4 nm and the actin layer-line at 5.92 run, respectively. In addition to the prominent actin layer-lines, a series of meridional and near-meridional reflections at, orders of 2 x 38.3 nm is seen up to the 24th order (3.2 nm).

STRUCTURAL

CHANGES

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FILAMENTS

IMh

maxima could not be measured precisely, but they fell in the range 0.02 bo 0.05 nm - ‘1, These feat*ures can be explained by assigning the reflections to TN molecules as will Iat: shown in Discussion, section (b). Ln support, of this assignment, the 2nd-order reflection at 38-8 nm corresponds to the meridional reflect’ion (at 38.5 nm) which haa been recognized as arising from TN in vertebrate muscle (Huxley & Brown. 1967: Rome et al., 1973). (b) Muscle in rigor (i) Equatorial

re$ection,s

Eight reflections were observed on the equator, and were indexed as 1,O; 1.1: 2.0: ?,l : 3.0; 2.2+3.1: 4,0 and 3,2+4,1 reflections from the hexagonal filament, lattice. .It tho sarcomere lengths of 5.0 to 5.5 pm, the spacing between the 1,O lattice planes was 53.1 to 54.5 nm. and the intensity of the 1,l reflection was st.ronger than that, of the 1 .O reflection. (ii) J~eridio?zal reJections assigned to the thick Jilaments Two meridional reflections, observed at 14.4 and 4.8 nm, were interpreted to be thrb 1st and 3rd orders of an axial repeat of 14.4 nm present in the thick filaments: these reflections were absent in the patterns from the muscles treated with myosinext,racting solution. They were much weaker than those observed in the resting state. (iii)

Layer-lines

assigned to actin

The axial patterns from the muscle in rigor (Fig. l(c) and (d)) showed the series of layer-lines which have been interpreted as arising from the helical arrangement ot iictin and TM molecules in other types of muscle (Huxley & Brown, 1967; Haselgrovr. 1972; Vibert et al., 1972; Parry & Squire, 1973). As shown in Table 2. all the layerlines fell very close to the orders of 2 x38.3 nm when their axial positions were measured at the intensity maxima in the off-meridional region (the 28th order. however: was a meridional reflection and its axial posit,ion was measured on tht, meridian). TABLE 2 Lnyer-lines

characteristic Order of 2 x 38.3 nm 1

2 4 7 9 11 13 15 26 28

of actin given by crab leg ,muscle in rigor

Axial

spacing cl (nm)

38.2 19.1 10.9 8.82 6.98 5.92 5.12 2.93 2.73

11 x 1 (nm)

76.4 76.4 76.3 76.7 76.8 77.0 76.8 76.2 76.4

The axial spacings were measured at the intensity maxima in the off-meridional m the case of the meridional reflection at I = 28. The values have probable errors 04%. The averrtge value of d x 2 is 76.6 nm.

region, except of approximately

Y.

196

MAfiDA,

I. MATSUBARA

AND

N. YAGI

The 2nd and 4th layer-lines had distinct intensity peaks on the meridian, and the 9th, llth, 13th and 15th layer-lines were extended onto the meridian. These features cannot be explained by the helical arrangement of actin and TM molecules. Neither random nor complete decoration of actin with myosin heads can account for these features (but see below). (iv) Reflections assigned to TN and crossbridges A prominent feature of the rigor pattern was a series of uniformly spaced reflections on the meridian and in the near-meridional region. These reflections, together with the meridional and near-meridional components of the 2nd, 4th, 9th, llth, 13th and 15th layer-lines assigned to actin, could be indexed as orders of 2 x 38.3 nm (Table 3). The lower-order reflections (2 5 9) were identified as the same reflections as were assigned to TN in the living resting pattern. However, comparison of the resting and rigor patterns (Fig. l(b) and (d)) indicates that these reflections are much stronger and seen up to higher orders in rigor. This suggests that in rigor muscle an additional structural feature contributes to these reflections. One possible explanation is that the myosin heads are attached to the thin filaments at regular intervals as is the case in insect flight muscle (Reedy, 1968) (see Discussion, section (c)).

TABLE 3 Meridional Order of 2 x 38.3 nm 1

27

3 4t 6 7 8 9 10 11t 1st 16t 17 18 19 20

w

22 23 24

and near-meridional

rejlections

Measured spacing d bm)

d x 1 (W

38.3 25.3 19.2 12.8 10.9 9.60 8.50 7.67 6.93 5.88 5.08 4.48 4.26 4.02 3.83 3.60 3.48 3.31 3.19

76.6 76.9 76.8 76.8 76.3 76.8 76.6 76.7 76.2 76.4 76.2 76.2 76.7 76.4 76.6 76.6 76.6 76.1 76.6

in crab leg muscle in rigor

intensity maximum the meridian

on

The 2nd, 4th, 6th and 8th orders correspond to the series of meridional reflections (et orders of 38.6 nm) assigned to TN-TM complex in glycerol-extracted Limulua muscle (Wray et al., 1974). Values have probable errors of approximately 0.4%. The average value of d x I is 76.6 nm. t These reflections might be linked to or superimposed on the a&in layer-lines at the seme axial level. 1 This reflection could be the 4th order of the thick filament periodicity (14.4 nm).

STRUCTURAL

CHANGES

IN

THIN

FILAMENTS

197

Some differences were noticed between the features of the even-order reflections and those of the odd-order reflections. The even-order reflections : (1) all the even-order reflections had intensity maxima on the meridian. (2) Certain even-order reflections (I = 12, 14, 16) were absent. This was likely to be due to a selection rule, as the reflections at the higher orders (I > 16) were present indicating that, t,he reflections did not simply fade out above t,he 12th order (SW Discussion, section (c)). The odd-order reflections : (1) all odd-order reflections except 1 = 21, 23 had intensity maxima in t’he nearmeridional region, and therefore looked as if they were split across the meridian ahhough t,hey had some intensity on the meridian. The radial positions of the intensity maxima could not be measured precisely, but. they fell in the range 0.01 to 0.03 nm-I. An intensity maximum was present on the meridian at the 21st order (3.6 nm), but t his might be due to the presence of the 4th-order reflect,ion of the 14.4 nm periodicity. The 23rd-order reflection was so faint that the radial posit,ion of the int,ensit,y maximum c*ould not be determined. (2) The refle&ons I = 1,5 were not visible. This, however, did not necessarily indicate that they were absent since they might not have been detected for the following reasons; t,he expected position of the lst-order reflection was in the region (of’ the intense central background scatter, and that of the 5th~order (15.3 nm) was close to the strong myosin reflection at 14.4 nm. When the glycerinated muscles were treated with myosin-extracting solution, t,htb myosin reflections disappeared completely. Most of the actin layer-lines also disappeared leaving an arced layer-line at about 5.9 nm, indicating that the t’hin filaments were disordered by dissolving the thick filaments. Among the meridional and Ilear-meridional reflect,ions at) orders of 2 x 38.3 nm, some meridional reflections a’t low orders (1 =m-2, 4, 6) survived the treatment although their int,ensibies were signific*antlJ- reduced. As the myosin heads were supposed to have been extract’ed, the survival of t*hc lower-order reflections suggests t’hat at least some of the reflect’ions i ndexiug on 2 x 38.3 nm are contributed to by TN molecules.

4. Discussion (a) Symmetry

of the actin

helix

Tn the patterns from muscles in rigor, all the layer-lines associated with actin could be indexed as orders of 2 x 38.3 nm (Table 2), indicating that the actin helix is integral. The “5.9 nm” layer-line, which gives the pitch of the genetic helix linking all subunits. occurred at 1 = 13, and the innermost meridional reflection, which gives the axial repeat of the actin subunit, occurred at 1 = 28. Such results indicate that each repeat of t’he actin helix (2 x 38.3 nm) contains exactly 28 subunits in 13 turns of the genetic helix (the helical symmetry: 28/13). The helix may be described alternatively as two helical strands (each of them with a pitch of 76.6 nm and 14 subunits per turn) caressing over each other at 38.3 nm intervals. Tn living resting muscles, we determined the crossover repeat of the long-pitch actin helices from the axial position of the innermost actin layer-line. The axial width of this layer-line was less than that in the patterns from other muscles (Vibert et al.. 1972), and so we could measure the spacing to within *O.Fi%. The crossover repeat

198

Y. MAfiDA,

I.

MATSUBARA

AND

N. YAGI

thus obtained was 36.6 nm which is intermediate between 35.4 and 383 nm, the values expected if the helix were 26112 and 28113, respectively. This indicates that in living resting muscle, the helix is intermediate between the two. Thus the present results have indicated that, when a living resting crab muscle goes into rigor, t’he symmetry of the actin helix changes. Such a change has not been detected before in the same muscle. However, previous measurements on various muscles, when reviewed in this light (O’Brien et al., 1975) suggest such differences between the actin helices in resting and rigor muscles. For instance. Huxley & Brown (1967) showed, based on their measurements on t’he separation of the “5.9 nm” and “5.1 nm” layer-lines, that the actin helix in frog skeletal muscle at rest has a nonintegral symmetry in between 26112 and 28/13 with a pitch of 2 x 36.0 to 2 x 37.0 nm. In frog muscle in rigor, their measurements suggested a small increase in the actin pitch, but this was not established firmly. Miller & Tregear (1972) showed that the actin helix in insect’ flight muscle in rigor has a symmetry of 28/13, and a crossover repeat of 38.5 nm. However, they did not determine t’he symmetry and the crossover repeat in relaxed muscle, since the axial broadness of the “5.9 nm” and “5.1 nm” layer-lines did not allow precise measurements. In paracrystals of reconstituted bhin filaments: the changes similar to those observed in the present study have been detected by Gillis & O’Brien (1975) using the optical diffraction technique applied to electron micrographs; they observed that, when the Ca2+ concentration of the medium suspending the paracrystals was changed from a low (<10m6 M) to a high ( >10A5 M) value, the symmetry changed from 26jlS to 28/13, and the crossover repeat from 35.5 to 38.4 nm. These changes were larger than those we found in the natural thin filaments; this was due t#o the fact that the parameters of the actin helix they obtained at the low Ca2+ concentration were different from the parameters we obtained at the living resting state. Such differences in the parameters might have been caused by the requirement of regular packing in paracryst’als; the packing may have obliged the helix to be integral. (b) Reflections assigned to

TN

in living

,resting muscle

A series of reflections indexing on 2 x 38.2 nm were observed on the meridian and in the near-meridional region in the living resting pattern. The following arguments indicate that some feat’ures of these reflections can be explained by assigning them to TN. (1) According to the model proposed by Ohtsuki (1974) (Fig. 2(a)), a pair of TN molecules are attached to the a&in-TM complex at intervals of about 38 nm. The pair of TN molecules are not aligned transversely, but are displaced axially to each other. The direction of this displacement alternates at 38 nm intervals, so the true crystallographic repeat of the TN arrangement along the thin filament is 2 x 38 nm. Such an arrangement would be expected to give rise to reflections at orders of 2 x 38 nm. In the actual patterns, the reflections were present at orders of 2x38.2 nm in agreement with this prediction. (2) Figure 2(b) shows TN molecules projected onto the thin filament axis in Ohtsuki’s model. The crystallographic repeat of TN in this projection is 38 nm. Therefore, the meridional reflections will occur only at the positions which can be indexed as orders of 38 nm. This means that, among the reflections indexed as orders of 2 x 38 nm, the meridional reflection will occur only at even orders. In agreement with this the

STRUCTURAL

CHANGES

IN THIN

FILAMENTS

I99

ActIn

! Ai

TN\

2*73nm

0

TM.

-T

38.3 nm

(a)

(b)

Fro. 2. The thin filament, structure in the rigor state. (a) Ohtsuki’s (1974) model for the arrangement of TN and TM. The true crystallographic repeat of the arrangement of TN is 2 x 38 nm (the precise value for crab leg muscle is 2 x 38.3 nm). The symmetry of the actin helix in this Kiguro is B/13. (b) The TN molecules projected onto the thin filament, axis. The crystallographic repeat in thie projection is 38 nm (the precise value: 38.3 nm). intensity maxima in the X-ray pattern were found on the meridian on1.y at even orders of 2 x 38.2 nm. Thus some features of the reflections under discussion can be explained by Ohtsuki’s model, supporting our assignment of these reflections to TN. Moreover, these reflections are unlikely to have arisen from myosin heads labelling actin (if any labelling were to occur in living resting muscle), since the axial position of the first meridional reflection (38.7 nm) does not agree with that of the innermost actin layer-line (36.6 nm). When these reflections are assigned to TN, we must conclude that, in living resting muscles, the axial repeat of the TN molecules on each TM strand does not agree with the cro,ssover repeat of the long-pitch actin helices. This means that, along each thin

200

Y.

MABDA,

I.

MATSUBARA

AND

N. YAGI

filament, the TN molecules form non-integral helices which would cause splitting of the off-meridional reflections (Franklin & Klug, 1955). However, the splitting expected in the present case is too tine to be resolved in our patterns. In Ohtsuki’s model the paired TN molecules are displaced axially from each other by 2.73 nm (Fig. 2). An alternative model which cannot be entirely excluded on the basis of Ohtsuki’s study (1974) would be the one with an axial displacement of 3x2-73 nm. Although such an arrangement of TN would also produce a series of reflections at orders of 2 x 38 nm, the meridional reflection at 1 = 4 would be in this case much weaker than the other meridional reflections. This does not agree with our observation that the reflection at 1 = 4 was as strong as other meridional reflections listed in Table 1. Therefore, we prefer the arrangement, with an axial displacement of 2.73 nm rather than 3 x 2.73 nm. (c) Rejiections

assigned

to TN and crossbridges in rigor

A series of reflections indexing on 2 x 38.3 nm was observed up to the 24th order on the meridian and in the near-meridional region in the rigor pattern. The following arguments indicate that these reflections are assignable to both TN and the myosin heads attached to the thin filaments at regular intervals. The main features of these reflections can be explained by assuming scattering centres arranged along the thin filament in the same manner as described for TN molecules in Figure 2. As was mentioned in the previous section, such an arrangement of scattering centres can explain the presence of the reflections at orders of 2 x 38 nm and the occurrence of intensity maxima on the meridian at the even orders. Furthermore, the absence of certain even-order reflections in the pattern (Results, section (c)) can be explained as follows. The projection of the scattering centres onto the thinfilament axis is assumed to be the same as in Figure 2; the axial displacement of the paired scattering centres is 2.73 nm, which corresponds to l/14 of their 38 nm repeat in the projection. The Fourier transform of this projection gives the structure factor on the meridian : PI’ = [l + exp(2&‘/14)] x [form factor of each scatterer] where 1’ represents the order of the 38 nm repeat (rather than the 2x38 nm repeat). This equation indicates that the intensity on the meridian will be extinguished at I’ = 7, and reduced at neighbouring values of 1’. This means that, when the reflections are indexed as orders of 2 x 38 nm, the meridional reflection will not be seen at the 14th order, and neighbouring reflections will be weak. In agreement with this prediction, no reflections were observed at the 12th, 14th and 16th orders in the actual pattern. Apparently the series of reflections under discussion can be assigned to TN as is the case with the identical series of reflections in the living resting pattern. However, if the reflections are attributed solely to TN, it becomes difficult to explain the difference between the resting and rigor patterns; the reflections are stronger and seen up to higher orders in the rigor pattern. To explain this we must assume a rather drastic change in the orientation of TN so that it is elongated longitudinally in the resting state and transversely in rigor. Such a drastic change is unlikely since no marked change has been noticed in the intensity of the meridional reflection (at 38.5 nm) assigned to TN when frog skeletal muscle goes into rigor (Huxley & Brown, 1967). Alternatively these reflections in the rigor patterns can be assigned to crossbridges. In rigor muscle the actin periodicity agrees with the periodicity of the scatterers

STRUCTURAL

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giving rise to the series of reflections. This makes it possible to assign the reflections to the myosin heads attached to each strand of long-pitch actin helices at a repeat of seven actin subunits; such an arrangement of myosin heads agrees with that of the scattering centres described above. A similar type of regular attachment of myosin heads has been found in insect flight muscle and attributed to the well-ordered structure of the myofilament lattice in which the thin filaments are themselves arranged in a helical manner round each thick filament (Reedy, 1968; Miller $ Tregear, 1972). It has been argued that myosin heads attach to the thin filaments only when they have the right azimuthal relationship to each other (Squire, 1972; Offer & Elliott, 1978). This leads to formation of crossbridges at regular, actinrelated intervals in well-ordered muscles. The crab muscle shows a well-ordered myofilament lattice when viewed with an electron microscope (Ma&da, manuscript in preparation), suggesting that it has the structural features necessary for a regular arrangement of crossbridges. The difference between the resting and rigor patterns can be best explained by attributing the intensities of these reflections in the rigor pattern to myosin heads attached to the thin filament, since such attachment is supposed to be abolished in resting muscle reducing the intensities. Preliminary calculations based on models of myofilaments have supported the idea that in general the regular attachment, of myosin heads to the thin filaments will contribute to these reflections. However, we do not consider that the reflections in the rigor pattern are assignable solely to the crossbridges, as some low-order reflections survived the treatment with the myosinextracting solution; there must be some contribution of TN to the reflections. Also the presence of the relatively weak low-order reflections in the resting pattern makes it very likely that TN contributes to these reflections in t’he rigor pattern. We are grateful to Professors S. Ebashi, M. Endo and F. Oosawa for advice and encouragement, and to Drs Pauline Bennett, Arthur Elliott, Ed O’Brien, Gerald Offer and Mr Roger Starr for helpful comments on earlier drafts of this paper. REFERENCES Ebaahi, S. (1972). Nature (London), 249, 217-218. Ebashi, S., Endo, M. & Ohtsuki, I. (1969). Quart. Rev. Biophys. 2, 351-384. Fatt, P. & Katz, B. (1953). J. Phyeiol. 120, 171-204. Franklin, R. E. & Klug, A. (1955). Actu Crystallogr. 8, 777-780. Gillis, J. M. & O’Brien, E. J. (1975). J. MOE. BioZ. 99, 445-459. Hanson, J. & Huxley, H. E. (1957). B&him. Biophys. Acta, 23, 250-260. Haselgrove, J. C. (1972). Cold Spring Harbor Symp. Quant. BioZ. 37, 341-352. Huxley, H. E. (1972). Cold Spring Harbor Symp. Qua&. Biol. 37, 361-376. Huxley, H. E. & Brown, W. (1967). J. Mol. BioZ. 30, 383-434. Miller, A. & Tregear, R. T. (1972). J. Mol. Biol. 70, 85-104. O’Brien, E. J., Gillis, J. M. & Couch, J. (1975). J. Mol. BioZ. 99, 461-475. Offer, G. BE Elliott, A. (1978). Nature (London), 271, 325-329. Ohtsuki, I. (1974). J. Biochem. 75, 753-765. Ohtsuki, I., Masaki, T., Nonomura, Y. & Ebashi, S. (1967). J. Biochem. 61, 817-819. Parry, D. A. D. & Squire, J. M. (1973). J. Mol. BioZ. 75, 33-55. Reedy, M. K. (1968). J. Mol. BioZ. 31, 155-176. Rome, E. M., Hirabayaahi, T. & Perry, S. V. (1973). Nature New BioZ. 244, 154-155. Squire, J. M. (1972). J. Mol. BioZ. 72, 125-138. Vibert, P. J., Haselgrove, J. C., Lowy, J. & Poulsen, F. R. (1972).J. Mol. RioZ. 71, 757-767. Wakabaywhi, T., Huxley, H. E., Amos, L. A. & Klug, A. (1975). J. Mol. BioZ. 93,477-497. Wray, J. S., Vibert, P. J. & Cohen, C. (1974). J. MOE. BioZ. 88, 343-348.