Meridional X-ray Diffraction Pattern Research Articles

A Synchrotron X-Ray Diffraction Study of Developing Chick Corneas

To study some ultrastructural aspects of developing chick corneas we performed a synchrotron x-ray diffraction analysis of 22 specimens obtained daily from developmental day 10 through day 19. Before day 12 of development in chicks we were unable to detect a meridional x-ray diffraction pattern from cornea. Neither were we able to record a first-order equatorial x-ray reflection at this time. Normally, these reflections are present in corneal x-ray patterns, arising from, respectively, the periodic axial electron density of fibrillar collagen and the lattice-like arrangement of the fibrils. By day 12 of development we could detect the third- and fifth-order meridional reflections (indicating increased amounts of collagen) and a first-order equatorial reflection (implying that more collagen was regularly arranged). The third- and fifth-order meridional reflections became more intense as the tissue matured, suggestive of a continued deposition of fibrillar collagen, and the scattering angle of the interfibrillar maximum increased, suggesting that regularly arranged collagen was becoming more closely packed with maturation. In embryonic chick corneas, the establishment of an orderly, fairly compacted matrix of collagen fibrils may be one of the main events underlying the acquisition of corneal transparency.

Interpretation of the X-ray diffraction pattern from relaxed skeletal muscle and modelling of the thick filament structure.

The first part of this paper is devoted to the model-building studies of our high resolution meridional X-ray diffraction patterns (in the region from 1/500 to 1/50 A-1) obtained from relaxed frog muscle. A one-dimensional model of thick filament was proposed which basically consists of two symmetrical arrays of 50 crossbridge crown projections. In the proximate and central zones of the filament the crossbridge crowns are regularly shifted with a 429 A period and appear as triplets with a 130 A distance between crowns, while the crowns in the distal parts of filament are regularly ordered with a 143 A repeat. The centre-to-centre distance between regions with crossbridge perturbations is 7050 A. The length of each crown projection is about 125 A. The model includes also (1) C-protein component represented in each half of the filament by seven stripes of about 350 A long and located 429 A apart, (2) a uniform density of filament backbone of about 1.5 micron length, and (3) 13 high density stripes in a central zone located with 223 A period. The final model explains very well the positions and intensities of the main meridional reflections. A three-dimensional model of crossbridge configuration is described in the second part of the work. The model was constructed by using the intensity profiles of the first six myosin layer lines of the X-ray pattern from stretched muscle and taking into account the crossbridge perturbations and the axial size of crossbridge crown obtained from the one-dimensional studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Phasing the meridional diffraction pattern of type I collagen using isomorphous derivatives

The meridional X-ray diffraction pattern of wet rat tail tendon contains information about the one-dimensional structure, or axial projection of electron density distribution of the type I collagen fibril. Using synchrotron radiation we have determined the intensities of the first 50 meridional X-ray diffraction reflections. The approach of isomorphous addition with reagents, selected using criteria of chemical reactivity, which label at fewer sites than the stains used in previous studies was applied to phase these 50 reflections to produce a one-dimensional electron density distribution map of a single D-repeat of the collagen fibril. This method is not model-dependent and thus constitutes the first unambiguous determination of the meridional phases of type I collagen.

Axial electron density of human scleral collagen. Location of proteoglycans by x-ray diffraction

The low angle meridional x-ray diffraction pattern from fresh human sclera was analyzed to ascertain if collagen-bound proteoglycans affect the axially-projected electron density distribution to the same extent as appears to occur in the cornea. The results showed that, unlike cornea, the electron density of the sclera is similar to that seen in rat tail tendon collagen. The proteoglycans were specifically stained using either Cuprolinic blue or Cupromeronic blue, both under critical electrolyte conditions. The tissue was then examined by electron microscopy and by low angle x-ray diffraction. The electron-optical observations suggested that proteoglycans associate with collagen near the d/e staining bands in the gap zone. A difference Fourier analysis from the x-ray results confirmed that these observations were not e.m. preparative artefacts and allowed a quantitative estimate to be made of the axial extent of the proteglycans in the wet tissue.

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle.

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.

The effect of the ATP analogue AMPPNP on the structure of crossbridges in vertebrate skeletal muscles: X-ray diffraction and mechanical studies.

Adenylylimidodiphosphate (AMPPNP), a nonhydrolysable analogue of ATP, has been used to arrest the crossbridge cycle of muscular contraction in one of its hypothetical intermediate states. Whole frog sartorius muscles were chemically demembranated, and it was found possible to cycle such skinned muscles reversibly between the relaxed and rigor states. The effect of binding of AMPPNP on the structure and spatial arrangement of the crossbridges of such muscles was studied using low-angle X-ray diffraction, with simultaneous recording of the mechanical effects, starting from the rigor state. Saturating concentrations of MgAMPPNP produce a characteristic decrease of about 50% in the original rigor isometric tension with a concomitant increase in muscle length by 0.13%. The equatorial X-ray diffraction pattern is modified in the following way: the lattice dimensions and the intensity of the (10) equatorial reflection do not change, while the intensity of the (11) equatorial reflection increases slightly. These observations of very small equatorial changes could be explained by assuming that in these muscles (as distinct from others such as rabbit psoas) the analogue does not produce a significant degree of detachment of crossbridges; that is, there are only AMPPNP-modified attached ones. The changes in the meridional X-ray diffraction pattern are more pronounced: the meridional reflection at 14.5 nm decreases in intensity, and the meridional reflection at 7.2 nm increases considerably: the intensity of all the actin-based off-meridional layer-lines decreases. There are no signs of the characteristic relaxed layer-lines, and the changes in the layer-line intensities are probably due to there being a single population of AMPPNP-modified attached crossbridges, rather than a mixture of attached and detached crossbridges. Thus the AMPPNP X-ray pattern, both equatorially and meridionally, is somewhat similar to the rigor one, indicating that most of the crossbridges remain attached. On the other hand, the fact that there are some changes in the layer-line intensities of the AMPPNP frog pattern, without the appearance of any signs of a relaxed equatorial pattern, indicates that the attached crossbridges are in a structural state that is different from rigor, one is not seeing, apparently, simply a mixture of rigor and relaxed states. Our tentative interpretation of this result is that there may be a structural change in the crossbridge near to the junction with S2, with less significant changes occurring in the parts of the crossbridge close to actin.

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae. Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found. A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.

Interpretation of the meridional X-ray diffraction pattern from collagen fibres in terms of the known amino acid sequence

A brief review is given of attempts to interpret the low-angle meridional X-ray diffraction patterns of tendon to yield the axially projected electron density of collagen fibrils. Both X-ray/electron microscope correlations and more conventional crystallographic methods have been used. For wet tendon, there have been two fundamental assumptions; either that those parts of the fibril which are positively stained in electron micrographs are intrinsically electron dense, or that the electron density is centrosymmetric. The validity of these assumptions is tested by synthesising models for the electron density based on the known amino acid sequence of collagen and the Hodge-Petruska scheme. Allowance is made for the effect of the water environment. These models are then Fourier transformed to give a set of calculated intensities for comparison with the observed low-angle meridional X-ray data. The calculated intensities are very sensitive to the conformation of the non-triple-helical terminal peptides (telopeptides) and to the D period. Difference Fourier syntheses are used to refine a model which gives the best agreement with the observed data whilst being both physically and chemically acceptable. The calculated phases of this model (model E) are then combined with the observed amplitudes to give a Fourier synthesis of the axially projected electron density in wet collagen fibrils. The implications of this result for the study of various metabolic and pathological situations in connective tissue are discussed.

Fine structure of the A-band in cryo-sections: The structure of the A-band of human skeletal muscle fibres from ultra-thin cryo-sections negatively stained

The technique of cryo-ultramicrotomy has been applied to the study of the ultrastructure of the A-band in vertebrate skeletal muscle. The tissue (open biopsies from human musculus tibialis anterior) was fixed briefly in glutaraldehyde, then treated with glycerol and rapidly frozen. Thin sections of frozen tissue were then negatively stained and viewed in the electron microscope. Preparations obtained in this way showed a marked improvement in preservation of certain types of order in the A-band. Most A-bands showed the same characteristic, but complex, pattern of transverse striations from the M-band to the outer edges of the A-band. Our purpose here is to give a general description of the cryo-sectioned A-band in terms of the relative spacings and densities of the various striations. A new nomenclature for different parts of the A-band is also suggested to facilitate future reference to the striations. The outstanding features of the striation pattern were: A pattern of three or five strong lines (M-bridges) in the M-band depending on fibre type. A pseudo-H-zone (here called the M-region) with a measured length of 1620 Å (±36 Å) and containing many closely spaced weak lines in addition to the M-bridge lines. Most of these were found to lie on one of two over-lapping periodicities of about 145 Å. Striations outside the M-region spaced about 140 Å to 150 Å and showing up clearly the myosin crossbridge repeat already known to be 143 Å. Different striations had different characteristic densities and their positions often appeared to be perturbed in a characteristic way from positions on a regular 143 Å repeat. The overall pattern therefore showed considerable complexity. A rapidly chaing pattern of striations everywhere except in the region of the A-band (here called the C-zone), where in other muscles C-protein is known to be located. But even in this region subtle changes in the pattern consistently appeared. Since the myosin repeat and the C-protein repeat have previously been reported to be identical (430 Å), no changes in pattern every 430 Å would be expected to occur. That changes do occur in cryo-sections must mean one of three things. Either the underlying packing arrangement of the myosin molecules must change throughout the C-zone, or actin filaments in the muscle must in some way cause the perturbation of the regular crossbridge arrangement, or the myosin packing is constant but the periodicities of myosin and C-protein are different. Because of the importance of the latter in the context of a possible length-determining mechanism in myosin filaments, we confine ourselves here to a preliminary description of the C-zone. An account of a continuing detailed study and analysis of the C-zone will be given elsewhere. That the complex pattern of transverse striations is representative of the axial structure in living muscle is indicated by the fact that optical diffraction patterns from whole cryo-sectioned A-bands are comparable to the meridional X-ray diffraction patterns from living vertebrate skeletal muscle (Huxley & Brown, 1967). This suggests that the cryo-sectioning technique has preserved for the first time the axial periodicities in the A-band and that this technique may help to bridge the gap between electron microscopy and X-ray diffraction.0115

Molecular tilting in dried elastoidin and its implications for the structures of other collagen fibrils

Wet elastoidin spicules (fish fin rays) yield low-angle meridional X-ray diffraction patterns which resemble those from tendons. However, when the spicule dries the meridian splits into the arms of a diagonal cross (sometimes only one arm appears). Of the possible explanations we reject shearing of the axial arrangement of molecules but confirm tilting. We suggest that, in three dimensions, the molecules are tilted at angles which vary from O o at the centre to some maximum value at the surface of the spicule, resembling torsion of the array of molecules. Molecular tilting probably occurs in other collagen fibrils.

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.

Relaxation of glycerinated muscle: Low-angle X-ray diffraction studies

Initially the general features and behaviour of glycerinated muscle in a relaxing medium were observed; experiments with glycerination techniques established the optimum conditions for relaxation. It was then possible to obtain consistently good relaxed specimens of glycerinated rabbit psoas muscle. Low-angle equatorial and meridional X-ray diffraction patterns were obtained from both rigor and relaxed specimens in the sarcomere length range 2.25 to 3.2 μm, and from relaxed specimens stretched to sarcomere lengths up to 4.4 μm. It was found that when a glycerinated muscle passes from the rigor to the relaxed condition at a fixed sarcomere length there is a large decrease in the intensity of the 1,1 equatorial reflection relative to the 1,0 reflection. The intensity ratio I(1,1) I(1,0) of relaxed glycerinated muscle is similar to (even slightly less than) that of the living resting muscle at the same sarcomere length. It is deduced that there is virtually complete detachment of the myosin projections from the actin filaments in the relaxed glycerinated muscle. There is also a change in the spacings of the equatorial reflections when a glycerinated muscle passes from the rigor to the relaxed condition. Over the whole sarcomere length range, 2.25 to 4.4 μm, the lattice spacing of relaxed glycerinated muscle varies with sarcomere length so that, as in living muscle, a constant lattice volume is maintained. In patterns from relaxed glycerinated muscle at sarcomere lengths above about 3.5 μm an additional equatorial reflection with spacing about 146 Å is observed; this is attributed to ordered arrays of actin filaments in the I-band. There is a radical change in the meridional pattern when a glycerinated muscle passes from the rigor to the relaxed condition: layer lines, of which the most prominent is at a spacing of about 365 to 385 Å, disappear; the spacing of the reflection corresponding to the myosin subunit repeat decreases by approximately 1% from 144.4 to 143.0 Å; other meridional reflections indexing on a repeat of 1155 (8 × 144.4) Å disappear and are replaced by reflections indexing on 429 (3 × 143.0) Å. These features demonstrate again that the myosin projections have been detached from the actin filaments in the transition from the rigor to the relaxed condition, and that the structural change in the myosin filaments which occurs when a living muscle passes into rigor is fully reversed when the rigor muscle is relaxed. It is concluded that the structure of relaxed glycerinated muscle, as determined from both equatorial and meridional X-ray diffraction data, is similar to that of the living resting muscle.