The axial repeats in paracrystals of light meromyosin and its complex with C-protein

We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.     

Electron microscopy and X-ray diffraction of a hexagonal net of light meromyosin

Light meromyosin, prepared by brief digestion of rabbit myosin, forms at low ionic strength tactoids with a 43 nm periodicity and open nets. These nets, when negatively stained, show strands intersecting at intervals of ~ 60 nm and at an angle of 120 ° to form hexagonal arrays (Huxley, 1963).By slow dialysis of light meromyosin from 0.35 to 0.1 m-KCl we have obtained large, highly ordered hexagonal nets, which we have subjected to structural analysis by electron microscopy of both negatively stained and sectioned material, and by X-ray diffraction. The net is a three-dimensional crystalline array whose overall shape is that of an oblate ellipsoid. Viewed down the short axis, a hexagonal appearance is seen. Analysis of other views of the net suggests that it has a simple layered structure, each layer consisting of a set of parallel strands of diameter about 10 nm. Each strand crosses over those in neighbouring layers at intervals of 64.4 nm and at an angle of 120 °, so that in the whole structure there is a 3-fold screw axis through each node of the net. A model for a strand is described in which light meromyosin molecules, ~ 100 nm in length, are arranged in an anti-parallel manner, each molecule having one end at a node of the lattice. If this end corresponds to the free end of the myosin tail, one of the interactions is similar to that found in type 1 segments of myosin rod (Harrison et al., 1971). The molecular packing within strands may be related to the packing of myosin tails in the bare zone of muscle thick filaments.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Crystalline structure of the gas vesicle wall from Anabaena flos-aquae.

The gas vesicles isolated from Anabaena flos-aquae have been studied by X-ray diffraction. Electron microscopy has previously shown that the gas vesicles are elongated shapes, with a thin wall having regular striations (ribs) at right-angles to the long axis. The X-ray diffraction pattern from a specimen of oriented, intact vesicles includes a number of sharp reflections which are attributed to regular structure in the plane of the wall. After correcting for the imperfect alignment of the long axes of the vesicles, the in-plane reflections are all seen to lie on a few, regularly spaced lines parallel to the long axis. This result shows for the first time that there are subunits regularly spaced along each rib, one subunit every 11 Å. The spacing of the in-plane reflections along each line is consistent with a rib periodicity of 46 Å. The 11 Å repeat, together with the 46 Å repeating distance from rib to rib and the average wall thickness of about 20 Å, define a volume for the subunit. Assuming a reasonable value for the density of the protein making up the wall, the molecular weight of the subunit indicated is about 8000 g/mol.The X-ray data also indicate that a large part of the protein is in the β-sheet conformation. In this structure there are parallel, or anti-parallel, polypeptide chains which are hydrogen-bonded to one another in a regular way to form a thin sheet. Assuming the wall contains β-sheet in two layers, one on top of the other and with the chains in each layer tilted at 35 ° to the long axis of the vesicle, we can explain a number of the X-ray observations: (1) oriented arcs with a Bragg spacing of 4.7 Å, which is the distance between the axes of neighbouring chains in each layer; (2) diffraction oriented in the direction of the chains at a spacing of 6 to 7 Å, which is the repeating distance of the dipeptide unit along the chain; (3) the 11 Å repeat, which is the repeating distance of pairs of chains along each rib; and (4) a broad band of diffraction at right-angles to the plane of the wall and centred at a spacing of 10 Å, which is a reasonable value for the distance between the mid-planes of the two sheets. Moreover, we can also find the remaining lattice parameter, the angle relating the centres of the subunits in neighbouring ribs. Thus the shortest line joining the centres makes an angle of 86 ° with the direction of the ribs.     

Banding and polarity of actin filaments in interphase and cleaving cells

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.     

PHOTOMOVEMENT RESPONSES OF PORPHYRIDIUM PURPUREUM1

The influence of light quality on positive phototopotaxis by the gliding, unicellular red alga Porphyridium purpureum was obtained using interference filters. Cells exposed to 3 × 10^?7 mol · m²· s^?1 of various wavelengths for 72 h showed maximum topotaxis at 420 and 440 nm. The lower threshold for positive, movement was approximately 5 × 10^?8 mol · m^?2· s^?1. Random movement occurred at nonactinic wavelengths, and no movement occurred in the dark. Cell motility appeared to be unaffected by light polarity, suggesting that the photoreceptor(s) for topotaxis and photokinesis are randomly oriented.     

Crystalline sheets of tropomyosin

In the presence of spermine tropomyosin forms sheets having two-dimensional crystallinity and tactoids. The most common form of sheet has cmm symmetry with a = 80 nm and b = 5 nm. The structure of this sheet has been solved in projection to a nominal resolution of 1.5 nm by combining data from electron diffraction and electron microscopy. Analysis of this pattern and that of rarely observed sheets having p2 symmetry (a = 40 nm, b = 5 nm and γ = 80 °) indicated that the cmm structure was formed by superposition of two p2 sheets. The tropomyosin molecules in each p2 sheet were arranged in rows directed along the p2 (0, 1) lattice lines, with all the molecules in one row having the same polarity and lying antiparallel to the molecules in adjacent rows. These rows associated in pairs, possibly by the supercoiling of the molecules in one row about those in the neighbouring row.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

A periodic structure in the 'pen' chitin of the squid Loligo vulgaris

The organization of the chitin-proteoglycan in Loligo vulgaris pen was examined ultrastructurally and related to the molecular order indicated by X-ray diffraction. There is a centrosymmetric striated repeat of 22 nm in the system which is based upon dark and light bands of unequal width. The banding is orientated perpendicular to the direction of the major molecular axis of the chitin fibres. The chitin molecules are laid down in sheets with a mutual, though irregular, twist to produce a laminated 'plywood' material.     

Closest packing of two-stranded coiled-coils as a model for the collagen fibril.

We propose that in the collagen fibril, the triple-helical molecules form two-stranded coiled-coils of period 5 × 670A?. Coiled-coils are packed on a tetragonal lattice and are axially staggered with ten in the unit cell (observed side 55A?) so that it carries the 670A?periodicity of the fibril. When nearest neighbours have opposing supercoil hands, the observed tetragonal lattice represents closest packing of two-stranded coiled-coils. This proposal is consistent with the row line spacings measured from the low angle X-ray diffraction pattern of tendon and explains the systematic absences and the two undisputed equatorial reflections. Unlike explanations for the diffraction pattern which invoke a five-stranded microfibril, our interpretation is consistent with its equatorial intensity distribution.     

Structural studies on porcine brain tubulin in extended sheets

Structural studies have been conducted on porcine brain tubulin, assembled into microtubule-related structures, by electron microscopy in conjunction with optical diffraction and image reconstruction techniques. By minimizing background noise and sample damage, we have improved the resolution on negatively stained samples, extending the data from the previous limit of a 42 Å layer line to an additional layer line at 21 Å. The new reflections confirm the basic surface lattice proposed from the earlier studies and extend the structural features that can be assigned to individual tubulin molecules. Data are obtained for microtubules in standard buffers for both the helical form and flat sheets of up to 13 protofilaments. When zinc is added to the preparations, sheets with more than 13 protofilaments are formed and the extended lattices provide more reflections on both the 42 Å and 21 Å layer lines, as well as the equator. The lattice in the presence of zinc differs considerably from the normal lattice, with adjacent protofilaments staggered by 21 Å, compared to the staggering of adjacent filaments of about 10 Å in the absence of zinc. There is also a distinct pairing of adjacent protofilaments in the zinc-induced sheets. Initial studies with the Unwin-Henderson method on unstained zinc-tubulin sheets suggest that the adjacent protofilaments may be related by a dyad axis, either perpendicular or parallel to the protofilament axes.     

On the tertiary structure of tropomysin tactoids.

Correlation of tropomyosin amino acid sequence with uranyl acetate stained magnesium salt tactoids has been carried out by a computer graphic technique. Contrary to some previous suggestions, the results seem to indicate that there is no chain stagger within the molecule and the molecules are aligned with 178 ± 2 residue overlap in antiparallel array. In addition, the molecules for one particular tactoid appear to have an end overlap of approximately 21 residues.     

Circular dichroism of the adenine and 6-mercaptopurine nucleotide complexes of heavy meromyosin

Circular dichroic CD spectra recorded below 250 nm indicate that myosin, heavy meromyosin (HMM), and subfragment-1 (S-1) contain 72, 58, and 32% α-helix. These percentages are consistent with the contention that heavy meromyosin and S-1 production is simply the result of partial and complete removal from myosin of its 95–100% helical tail. Further evidence that the globular heads are similar in the three proteins is the presence of four positive (near 299, 272, 265, and 259 nm) and two negative (near 290 and 283 nm) bands in the CD spectrum of all three. Complex formation of adenylyl imidodiphosphate and ADP with heavy meromyosin results in small changes in the 280- to 260-nm region of the circular dichroic spectrum. Production of the ATP hydrolysis steady state causes 50–60% increases in the ellipticities of the 259- and 283-nm bands, and a 50% decrease in the 272-nm band. Similar experiments using 6-thioinosine triphosphate show that the ellipticity of the nucleotide in the steady state is more than twice as large as that of the diphosphate complex. Since rotatory strength most commonly arises from the coupling of electronic transitions of neighboring chromophores, the results suggest that an aromatic residue (probably tryptophan) moves near the purine of the nucleotide upon hydrolysis to HMM1ADP·P (the steady-state complex) and then moves away during conversion to HMM·ADP·P (the post-steady-state complex). This relative movement between an amino acid side chain and the nucleotide may be part of an early stage of the mechanism by which hydrolytic energy is transduced to relative movement between the filaments of the myofibril.     

X-ray diffraction patterns from mammalian heart muscle

We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart.     

A 12 Å resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 Å resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 Å. The diffraction patterns changed dramatically at 150 Å; consequently, the low (less than 150 Å) and high (greater than 150 Å) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.     

In the mammalian eye type VI collagen tetramers form three morphologically different aggregates.

The organization of the aggregates occurring in the stroma: (1) of the murine and human cornea after incubation in an ATP acidic solution; (2) of surgically excised epiretinal membranes (ERM); and (3) of the trabecular meshwork of monkey eyes was investigated morphologically and immunocytochemically on thin section electron microscopy. Morphology. The aggregates in the cornea appeared as cross-banded fibrils. The bands were uniformly electron dense (single banded form); they were separated from each other by interbands consisting of a bundle of filaments emerging in cross section as small areas of randomly assembled dot-like structures. In the ERM, most of the aggregates stood out as heteromorphic cross-banded bodies showing dense bands with electron denser borders (double banded form) and interbands composed of longitudinally oriented, parallel sheets or laminae of amorphous material enclosing thin, similarly oriented filaments. These extended, thinner and double in number (since interlacing with similar components of the opposite sheet), into the pale central zone of the dense band. The aggregates of the trabecular meshwork were heteromorphic, had uniformly dense bands (single banded form as in the cornea), but their interbands displayed longitudinal sheets (as the ERM aggregates). Immunocytochemistry revealed type VI collagen in the three eye aggregates with gold particles preferentially localized at the interbands. The specificity of the antibodies used was tested by Western blot analysis of type VI collagen samples extracted from human placenta and on homogenates of human cornea. In conclusion, the results indicate that the tetramers of type VI collagen may aggregate differently into structures with distinct supramolecular arrangements. These are illustrated in schematic drawings.     

Conformations and interactions of pectins: I. X-ray diffraction analyses of sodium pectate in neutral and acidified forms

X-ray diffraction patterns of uniaxially oriented, polycrystalline fibers of neutral sodium pectate can be indexed on the basis of an orthogonal unit cell with dimensions a = 0.84 nm, b = 1.43 nm, c (fiber axis) = 1.34 nm, which contains trisaccharide fragments of two polygalacturonic chains of opposite sense. The polysaccharide chains have 3₁ screw symmetry but are arranged in a lattice that has space group symmetry P2₁ (unique axis b). There are three sodium ions in each crystal asymmetric unit. They are all octahedrally co-ordinated to oxygen atoms of the galacturonan chains or of water molecules. Every oxygen atom is involved also in at least one hydrogen bond. Sodium pectate can be partially converted to pectic acid whose polysaccharide chains preserve the 3₁ pectate conformation, are packed in an orthogonal unit cell also with P2₁ symmetry but with quite different dimensions a = 0.99 nm, b (unique 2₁ axis) = 1.23 nm, c (fiber axis) = 1.33 nm. In this lattice, the polygalacturonic acid chains form corrugated sheets in which alternate molecules have opposite sense and are extensively hydrogen-bonded through their carboxyl groups.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle.

X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al. (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z-disc lattice that correlated very closely with the X-ray results. It is suggested that the Z-disc structure is the major source of this nonindexible reflection.     

The crystalline structure of collagen fibrils in tendon.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with $\text{b = 75.5}\text{A}\text{?}$, β = 93 °, $\text{a =}\text{b}\text{sin}\text{β}$, a = 90 °, $\text{c = n × 668}\text{A}\text{?}$, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.