Intensities Of Layer Lines Research Articles

Rapid small-angle X-ray diffraction of a tonically contracting molluscan smooth muscle recorded with imaging plates

Small-angle X-ray diffraction patterns from the anterior byssus retractor muscles of Mytilus edulis contracting tonically in response to stimulation with acetylcholine were recorded in a 30 s exposure with synchrotron radiation and a high-sensitivity X-ray area detector called an imaging plate. The 190 Å layer line from the thin filaments increased in intensity with increase in tonic tension up to 6 x 104 kg m−2. Above this value, the layer-line intensity remained almost constant and comparable to that for a contracting skeletal muscle, indicating that the same structural changes of the thin filaments occur in both muscles.

Theoretical Fraunhofer light diffraction patterns calculated from three-dimensional sarcomere arrays imaged from isolated cardiac cells at rest

Sarcomere striation positions have been obtained throughout the volumes of calcium-tolerant resting heart cells by direct computer interfaced high-resolution optical imaging. Each sarcomere position is stored in a three-dimensional (3-D) matrix array from which Fraunhofer light diffraction patterns have been calculated using numerical methods based on Fourier transforms. Diffraction patterns have been calculated from heart cell data arrays oriented normal to a theoretical laser beam. Twelve characteristic features have been identified and described from these diffraction patterns that correlate to diffraction phenomena observed from both cardiac and skeletal muscle. This numerical approach provides the means to directly assess diffraction pattern formulation, the precision of layer line angular separation, layer-line intensity and angular asymmetries, line widths and fine structures in terms of the known diffracting source structures. These results confirm that theoretical calculations can predict real muscle diffraction patterns and their asymmetries.

The variable twist of actin and its modulation by actin-binding proteins.

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.

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.

The Fourier transform of actin and other helical systems with cumulative random angular disorder

A general class of helical disorder exists which can be described by cumulative random angular motions of subunits. This disorder affects layer-line intensities and widths by a factor proportional to n2, the square of the order of the layer line. The result explains several features of actin and polytetrafluoroethylene (Teflon) transforms, and may be relevant to other helical systems.

Coordinated use of isomorphous replacement and layer-line splitting in the phasing of fiber diffraction data

In order to calculate electron density maps from fiber diffraction patterns, the terms which superimpose because of cylindrical averaging of the pattern must be separated and their phases determined. This can be done using a technique analogous to the isomorphous replacement method of protein crystallography, but large numbers of heavy-atom derivatives are required. Because of the cylindrical averaging, the number of derivatives increases with increasing resolution. This paper describes a method for measuring the fine splitting of layer lines which occurs when a helical structure repeats approximately, but not exactly, in a given number of turns, and for using this as a source of phase information. The amount of phase information obtainable from each heavy-atom derivative is theoretically doubled since differences in both layer-line intensity and apparent layer-line position can be used, and this substantially increases the resolution attainable with a limited number of derivatives. The method is used to calculate an electron density map of tobacco mosaic virus at 6.7 Å resolution using only two derivatives instead of the four which would have been required using previously available methods.

The agarose double helix and its function in agarose gel structure

Agarose and eight different derivatives carrying O-methyl, O-sulphate, O-hydroxyethyl or O-carboxyethylidene substituents in various positions were studied by optical rotation, X-ray diffraction and computerised molecular model building methods. All samples showed essentially the same order-disorder transition during gel-sol interconversion. In addition, all the samples that could be made into oriented films or fibres gave X-ray diffraction diagrams corresponding to a common molecular structure. A double helix model for this structure is proposed that has the 0.95 nm axial periodicity observed and a calculated cylindrically averaged Fourier transform in good agreement with the observed (continuous) layer line intensities. Each chain in the double helix forms a lefthanded 3-fold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance. This model accounts for the sign and magnitude of the optical rotation shift that accompanies the sol-gel transitions and is sterically accessible to each of the various substituted forms. The relationship between agarose gel properties and the double helix is discussed and the structure compared with i-carrageenan.