Fiber Diffraction Data Research Articles

The location of water around the DNA double-helix

The diffractometer D19 at the ILL has been used to locate water around the DNA double-helix in fibres of the polynucleotide poly[d(A-T)].poly[d(A-T)]. Neutron fibre diffraction data were recorded to a resolution of 3 Å first when the DNA was surrounded by D 2O and second when it was surrounded by H 2O.

The double-helical nature of the crystalline part of A-starch

A new three-dimensional structure of the crystalline part of A-starch is described in which the unit cell contains 12 glucose residues located in two left-handed, parallel-stranded double helices packed in a parallel fashion; four water molecules are located between these helices. Chains are crystallized in a monoclinic lattice with a = 2·124 nm, b = 1·172 nm, c = 1·069 nm and γ = 123·5 °, the c axis being parallel to the helix axis. Systematic absences are consistent with the space group B2. The structure was derived from joint use of electron diffraction of single crystals, X-ray powder patterns decomposed into individual peaks and previously reported X-ray fibre diffraction data after adequate re-indexing. The repeating unit consists of a maltotriose moiety where the glucose residues have the 4C 1 pyranose conformation and are α(1 → 4) linked. The conformation of the glycosidic linkage is characterized by torsion angles (Φ, ψ) which take the values (91·8,−153·2), (85sd7−145·3) and (91·8,−151·3); all the primary hydroxyl groups exist in a gauche-gauche conformation. There are no intramolecular hydrogen bonds. Within the double helix, interstrand stabilization is achieved without any steric conflict and through the occurrence of O (2)O (6) type hydrogen bonds. The present structure is consistent with both physicochemical and biochemical aspects of the crystalline component of the cereal starch granules.

Difference Fourier syntheses in fiber diffraction

Theory applying to difference Fourier syntheses from fiber diffraction data is developed, including the calculation of expected peak heights and noise levels. The signal-to-noise ratio in fiber diffraction difference maps is much lower than in crystallography, because of the multi-dimensional nature of fiber diffraction data, but it is shown by means of examples from tobacco mosaic virus that high-order difference syntheses, for example using coefficients analogous to crystallographic 6Fobs - 5Fcalc, can clearly reveal differences between an observed structure and a model. 'Omit' maps, calculated from models by omitting a region under particular scrutiny, are of limited use in fiber diffraction, but maps calculated from hybrid coefficients derived from both full and partial models have some applications.

Microtubule structure at 18 Å resolution

A model for the structure of microtubules at a resolution of 18 Å (1 Å = 0.1 nm) is described, based on X-ray fiber diffraction data from hydrated reassembled calf brain microtubules. The model was derived by an iterative solvent flattening refinement procedure, with initial phases based on those determined by electron microscopy. The major microtubule surface grooves are those defining the protofilaments, which form a hollow cylinder of maximum diameter 300 Å. Strong electron density fluctuations in the microtubule wall are interpreted as evidence for a domain structure within the tubulin subunit. The arrangement of domains is such that the tubulin molecule could be quite flexible at the domain connections; thus, slight changes in this arrangement could account for the unusual polymorphism of tubulin assemblies.

Pf1 Inovirus: Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 Å resolution and single isomorphous replacement data to 5 Å resolution

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 Å resolution in the meridional direction on 60 layerlines that are resolved to 4 Å in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of α-helix with its axis at an angle of about 20° to the axis of the virion. The α-helix subunit curves around the virion axis by about 1 6 turn, and decreases from about 27 Å radius to about 13 Å radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour α-helical subunits are about 10 Å apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

The structure of the double-stranded RNA pentamer 5'(CACAG) . 5'(CUGUG) determined by nuclear Overhauser enhancement measurements: interproton distance determination and structure refinement on the basis of X-ray coordinates.

The structure in solution of the duplex RNA pentamer 5'(CACAG) . 5'(CUGUG), comprising the stem of the T psi C loop of yeast tRNAPhe, has been investigated by means of one- and two-dimensional nuclear Overhauser enhancement measurements. All non-exchangeable base and sugar proton resonances with the exception of the H5'/H5" sugar resonances are assigned in a sequential manner. From the relative intensities of the cross-peaks obtained in the pure-phase absorption two-dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A-type conformation in solution. Cross-relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effects, principally by one-dimensional measurements. The structure of the RNA pentamer is then refined by restrained least-squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propeller twist, base roll and base tilt angles.

The structure of collagen.

A knowledge of the structure of collagen fibrils is important for any rational discussion of the occurrence and treatment of fibrosis. The different genetic types of collagen, and the structure of the triple-helical molecule as refined from X-ray fibre diffraction data, are described. The problem of determining molecular arrangement in native tissues is discussed. The various models proposed for the molecular arrangement of type I collagen are compared and an account is given of the quasi-hexagonal model. A detailed analysis of the X-ray diffraction patterns from native type I collagen fibres is used to provide a quantitative description of the quasi-hexagonal model. Parameters such as molecular positions, azimuthal orientation and axial shift can be estimated from the diffraction patterns. These parameters refer to the helix main-chain. Side-chain conformations can then be built in by molecular graphics and the predicted X-ray pattern for the complete model compared with the observed pattern.

Base-stacking interactions in double-helical DNA structures: experiment versus theory.

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fibre diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.

Use of axial Pattersons in Assessment of Compatibility of alternative B-DNA structures with fibre X-ray data

Abstract Results are reported for an analysis of the compatibility of various models for duplex DNA with available X-ray data for moist DNA fibres using axial Pattersons. The models considered are the Arnott refined double helix, the Levitt modified double helix and a stereochemically refined version of the side-by-side structure. This relatively simple test of gross structural features circumvents difficulties associated with the comparison of models based on determinations of atomic positions from the limited X-ray data available from paracrystalline samples. The advantage of the axial Patterson is that it is calculable directly from the fibre diffraction data, involves no assumptions about the molecular symmetry of the sample and is interpretable in terms of the gross molecular structure. While the results are not definitive they do enable the range of structural possibilites for duplex DNA to be explored.

Tobacco mosaic virus: interactions between RNA and protein determined by refinement against fibre diffraction data and equilibrium binding studies

Tobacco mosaic virus: interactions between RNA and protein determined by refinement against fibre diffraction data and equilibrium binding studies

Solvent-induced distortions and the curvature of alpha-helices.

The alpha-helix defined in 1951 by Pauling et al. on the basis of model building and X-ray fibre diffraction data has 3.65 residues per turn (n) achieved with planar peptides, torsion angles of phi = -48 degrees and psi = -57 degrees and hydrogen bonds which are close to linear. Although X-ray analyses of proteins have confirmed the general correctness of the model for the helix, recent high resolution (1.7-1.0 A) diffraction studies have shown that the parameters described by Pauling et al. and later by Perutz and Arnott and Wonacott are not a good description of the alpha-helices in globular proteins, where the mean values of phi, psi are usually close to -63 degrees, -42 degrees. Here we show that these values arise as a mean of two significantly different classes in amphipathic helices depending on whether the peptide carbonyl oxygen is hydrogen bonded to a solvent or polar side-chain atom. The hydrogen bonds made by the hydrophilic carbonyls to the NH groups within helices are longer and less linear than those involving hydrophobic carbonyls. We also show that these effects are associated with a significant curvature of helices in globular proteins. For example, the alpha-helix in avian pancreatic peptide (aPP) has a radius of curvature of approximately 70 A. These results are of significance in the packing of helices in fibrous and globular proteins, in the calculation of their dipole moments, solvent accessibilities and internal energies, and in the theoretical estimation of spectroscopic properties such as circular dichroism and Raman scattering.

Structure of D-DNA: 8-fold or 7-fold helix?

We have shown that both right- and left-handed uniform helical models (RU and LU models) could be built to give satisfactory agreement with the fibre diffraction data of poly[d(I-C)] in the D-form. Atomic coordinates of these two models are reported in the present work. Molecular transforms of these two models, as well as of the recently published Hoogsteen base-paired 7-fold helical structure of Drew and Dickerson, are given. In view of the work of Drew and Dickerson, attention is drawn to the presence of clear 004 and 008 reflections in the diffraction patterns of poly[d(I-C)] and poly[d(A-T)]. The available data strongly suggest an 8-fold helical structure for the D-form of DNA.

Maximum-entropy calculation of the electron density at 4 A resolution of Pf1 filamentous bacteriophage.

A 4 A electron-density map of Pf1 filamentous bacterial virus has been calculated from x-ray fiber diffraction data by using the maximum-entropy method. This method produces a map that is free of features due to noise in the data and enables incomplete isomorphous-derivative phase information to be supplemented by information about the nature of the solution. The map shows gently curved (banana-shaped) rods of density about 70 A long, oriented roughly parallel to the virion axis but slewing by about 1/6th turn while running from a radius of 28 A to one of 13 A. Within these rods, there is a helical periodicity with a pitch of 5 to 6 A. We interpret these rods to be the helical subunits of the virion. The position of strongly diffracted intensity on the x-ray fiber pattern shows that the basic helix of the virion is right handed and that neighboring nearly parallel protein helices cross one another in an unusual negative sense.

Structure of the filamentous bacteriophage, Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27 5 helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.

Left-handed helices for DNA: studies on poly[d(I-C)

Earlier, we showed that, for the D form (n = 8 and h = 3.03 A, where n is number of nucleotide units per turn and h is height per nucleotide unit) of poly[d(A-T)], both right- and left-handed double helical models are stereochemically satisfactory and give good agreement with the observed fiber diffraction data. It was also noted that the conformations of the right- and left-handed D-DNA models are very similar to those of the right- and left-handed B-DNA models. This observation was consistent with the D leads to B transition in the solid phase. As a continuation of our earlier studies, we have carried out similar experiments with poly[d(I-C)]. We could obtain a crystalline D-form pattern (n = 8, h = 3.13 A) of the fiber at 75% relative humidity (r.h.); the hydrated (r.h. approximately equal to 95%) form of the same fiber gave the classical B-form pattern (n = 10, h = 3.40 A). In the present report, we show that both right- and left-handed double-helical models are consistent with the fiber diffraction data of poly[d(I-C)] in the D-form. Theoretical energy calculations also suggest that the right- and left-handed B- and D-DNA models are almost equally stable. Hence, we conclude that the right- and left-handed double-helical models of poly[d(I-C)] in a given form (B or D) are equally likely and that the fiber diffraction data do not permit discrimination.

The use of continuous diffraction data as a phase constraint. II. Application to fiber diffraction data

The properties of intensity data in fiber diffraction patterns are considered with the aim of developing strategies for structural studies of helical macro-molecular assemblies. A quantitative calculation is made of the number of independent intensity measurements in a fiber diffraction pattern and the number of degrees of freedom in an image of a helical object. These calculations are based on sampling theory in cylindrical coordinates. Several novel schemes for sampling intensities in a fiber diffraction pattern are demonstrated. A class of Patterson-like functions are introduced that may be useful in determining the helical symmetry of a particle from diffraction data. The use of these results in determining the significance of details of structural models based on fiber diffraction data is discussed.

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.

A need for re-examination of the fibre diffraction data of A-DNA

A-DNA pattern, obtained using a flat plat camera, was indexed by Fuller et al 6 on the basis of a c-face centred monoclinic cell with a = 22.24 Å, b = 40.62 Å, c = 28.15 Å and β = 97.0°. A precession photograph of A-DNA which gives an undistorted picture of the lattice, showed that the unit cell parameters as given by Fuller et al were not quite correct. The precession photograph showed a strong meridional reflection (R = 0.00 Å −1) on the 11th layer line. But the occurrence of the meridional reflection on the 11th layer line could not be explained on the basis of the cell parameters given by Fuller et al 6 ; using those cell parameters the reflection which comes closest to the meridian on 11th layer line is at R = 0.025 Å −1. However, a simple interchange of a and b values accounted for the meridional reflection on 11th layer line. The corrected cell parameter refined against 28 strong spots are a = 40.75 Å, b = 22.07 Å, c = 28.16 Å and β = 97.5°. In the new unit cell of A-DNA, the packing arrangement of the two molecules is different from that in the old one. Nonetheless, our earlier contention is again reaffirmed that both right and left-handed A-DNA are stereochemically allowed and consistent with the observed fibre pattern.

Polymerization of α,α‐disubstituted β‐propiolactones and lactams, 12. Properties and crystalline structure of poly(β‐propiolactam)s

AbstractA series of α,α‐disubstituted β‐propiolactams (substituted nylon 3 polymers) was prepared from non‐optically active lactams. Their thermal characteristics, densities, and x‐ray fiber diffraction patterns were recorded. One of the α‐substituents was always a methyl group while the other was either CH3, C2H5, n‐C3H7, or n‐C4H9. The melting point for the dimethyl substituted polymer was 268°C; the others were respectively 76, 74, and 72°C. X‐ray fiber diffraction data on all four samples yielded the same fiber repeat of ≈8,4 Å. A minimum energy based conformational analysis for the α,α‐dimethyl derivative indicates that two conformations correspond to this repeat, each of which is a 21 helix.

Protein‐RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-chain derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.