Fiber Diffraction Patterns Research Articles

MuscleX: data analysis software for fiber diffraction patterns from muscle.

MuscleX is an integrated, open-source computer software suite for data reduction of X-ray fiber diffraction patterns from striated muscle and other fibrous systems. It is written in Python and runs on Linux, Microsoft Windows or macOS. Most modules can be run either from a graphical user interface or in a `headless mode' from the command line, suitable for incorporation into beamline control systems. Here, we provide an overview of the general structure of the MuscleX software package and describe the specific features of the individual modules as well as examples of applications.

Using Multiscale Simulations as a Tool to Interpret Equatorial X-ray Fiber Diffraction Patterns from Skeletal Muscle.

Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel "forward problem" approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick-thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle.

The 3D structure of fibrous material is fully restorable from its X-ray diffraction pattern.

X-ray fiber diffraction is potentially a powerful technique to study the structure of fibrous materials, such as DNA and synthetic polymers. However, only rotationally averaged diffraction patterns can be recorded and it is difficult to correctly interpret them without the knowledge of esoteric diffraction theories. Here we demonstrate that, in principle, the non-rotationally averaged 3D structure of a fibrous material can be restored from its fiber diffraction pattern. The method is a simple puzzle-solving process and in ideal cases it does not require any prior knowledge about the structure, such as helical symmetry. We believe that the proposed method has a potential to transform the fiber diffraction to a 3D imaging technique, and will be useful for a wide field of life and materials sciences.

Heating Wheat Gluten Promotes the Formation of Amyloid-like Fibrils.

Amyloid fibrils (AFs) are highly ordered nanofibers composed of proteins rich in β-sheet structures. In this study, the impact of heating conditions relevant in food processing on AF formation of wheat gluten (WG) was investigated. Unheated and heated WG samples were treated with proteinase K and trypsin to solubilize the nonfibrillated protein, while protein fibrils were extracted with 0.05 M sodium phosphate buffer (pH 7.0) from the undissolved fraction obtained by the same enzymatic treatment. Conditions (i.e., heating at 78° for 22 h) resembling those in slow cooking induced the formation of straight fibrils (ca. 700 nm in length), whereas boiling WG for at least 15 min resulted in longer straight fibrils (ca. 1–2 μm in length). The latter showed the typical green birefringence of AFs when stained with Congo red. Their X-ray fiber diffraction patterns showed the typical reflection (4.7 Å) for inter-β-strand spacing. These results combined with those of Fourier transform infrared and thioflavin T spectroscopy measurements validated the identification of β-rich amyloid-like fibrils (ALFs) in dispersions of boiled WG. Boiling for at least 15 min converted approximately 0.1–0.5% of WG proteins into ALFs, suggesting that they can be present in heat-treated WG-containing food products and that food-relevant heating conditions have the potential to induce protein fibrillation.

Reconstruction of Real-space 3-D Structure from X-ray Fiber Diffraction Pattern: Application to Muscle Protein Filaments

An X-ray diffraction pattern of an object is the Fourier transform of its electron density distribution. By applying a reverse Fourier transformation to the diffraction pattern, the original structure of the object should be restored. However, it is difficult to do this, because of the loss of phase information, which is indispensable for restoring the original structure. In the case of fiber diffraction pattern, it is even more difficult to restore the original structure, because the diffraction pattern is rotationally averaged around the fiber axis and the angular information is lost in addition to the phase information. Here we demonstrate that from high-quality fiber diffraction patterns, the non-rotationally-averaged real-space 3-D structure of the fiber can be restored without the need of phase information. The algorithm is simply to find one atom by one, in a 3-D space, to generate a set of atoms that do not contradict the recorded diffraction pattern. The algorithm is distinct from the iterative error reduction, which is commonly used in coherent diffractive imaging. We applied this technique to the protein filaments in muscle. The non-integral helix of the actin filament is readily restored. The multi-start integral helix of the myosin filament is more difficult to restore, but it is restorable anyway. This technique should be applicable, not only to biological filaments, but also to other materials, such as synthetic polymer filaments.

Laser diffraction images for determination of natural and man-made fibers

Differentiating between textile fibers is usually done by destroying chemical methods or expensive optical techniques. Another possibility was suggested earlier, utilizing the diffraction patterns of single fibers. Tests of a few man-made and natural fibers have revealed the principle capability of this method to distinguish between wool, cashmere and chemical fibers. Here we report on a more sophisticated setup, allowing for faster measurements, and a broader study of more than 30 different fibers, giving rise to the broad spectrum of possible diffraction patterns and correlating some general differences in these optical examinations with differences of the fiber morphologies.

Structure of Polyacrylonitrile Fibers Produced from N-Methylmorpholine-N-Oxide Solutions

Structural and morphological evolution of polyacrylonitrile (PAN) samples from starting powder of a ternary copolymer to fibers produced from concentrated solutions of PAN in N-methylmorpholine-N-oxide (NMMO) was studied using x-ray diffraction for the first time. X-ray exposures in transmission and reflection geometries allowed the structures of outer and inner parts of the PAN fibers to be differentiated. It was shown that a shell—core structure formed in the precipitation bath during fiber spinning. A comparison of x-ray diffraction patterns of fibers spun using the NMMO process and industrial samples spun from DMSO and aqueous sodium thiocyanate solutions did not reveal fundamental structural differences.

Hierarchical architecture of spider attachment setae reconstructed from scanning nanofocus X-ray diffraction data.

When sitting and walking, the feet of wandering spiders reversibly attach to many surfaces without the use of gluey secretions. Responsible for the spiders' dry adhesion are the hairy attachment pads that are built of specially shaped cuticular hairs (setae) equipped with approximately 1 µm wide and 20 nm thick plate-like contact elements (spatulae) facing the substrate. Using synchrotron-based scanning nanofocus X-ray diffraction methods, combining wide-angle X-ray diffraction/scattering and small-angle X-ray scattering, allowed substantial quantitative information to be gained about the structure and materials of these fibrous adhesive structures with 200 nm resolution. The fibre diffraction patterns showed the crystalline chitin chains oriented along the long axis of the attachment setae and increased intensity of the chitin signal dorsally within the seta shaft. The small-angle scattering signals clearly indicated an angular shift by approximately 80° of the microtrich structures that branch off the bulk hair shaft and end as the adhesive contact elements in the tip region of the seta. The results reveal the specific structural arrangement and distribution of the chitin fibres within the attachment hair's cuticle preventing material failure by tensile reinforcement and proper distribution of stresses that arise upon attachment and detachment.

Three-dimensional double helical DNA structure directly revealed from its X-ray fiber diffraction pattern by iterative phase retrieval.

Coherent diffraction imaging (CDI) allows the retrieval of an isolated object's structure, such as a macromolecule, from its diffraction pattern. CDI requires the fulfillment of two conditions: the imaging radiation must be coherent and the object must be isolated. We discuss that it is possible to directly retrieve the molecular structure from its diffraction pattern, which was acquired neither with coherent radiation nor from an individual molecule. This is provided that the molecule exhibits periodicity in one direction, as in the case of fiber diffraction. We demonstrate that, when we apply iterative phase retrieval methods to a fiber diffraction pattern, the repeating unit; that is, the molecule structure, can directly be reconstructed without any prior modeling. For example, we recover the the DNA double helix's structure in three-dimensions from its two-dimensional X-ray fiber diffraction pattern, Photograph (Photo) 51, which was acquired in Raymond Gosling and Rosalind Franklin's famous experiment at a resolution of 3.4 Å.

Effects of Myosin Inhibitors on the X-Ray Diffraction Patterns of Relaxed and Calcium-Activated Rabbit Skeletal Muscle Fibers

Two potent myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS) and blebbistatin, suppress the contractile force of skeletal muscle fibers at micromolar concentrations. To gain insights into the mechanism of inhibition by these chemicals, we studied their effects on the X-ray diffraction patterns from rabbit skeletal muscle fibers under relaxing and activating conditions. At 100 µM, both inhibitors almost completely suppress the contractile force, but still substantial changes were observed in the diffraction patterns upon calcium activation. (1) The 2nd actin layer line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a high concentration, myosin heads leave the helix on the myosin filaments and approach the actin filaments. Interestingly, the d1,0 spacing of the filament lattice remains unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.

Effects of myosin inhibitors on the X-ray diffraction patterns of relaxed and calcium-activated rabbit skeletal muscle fibers.

We studied the effect of myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS), blebbistatin, and butanedione monoxime (BDM) on X-ray diffraction patterns from rabbit psoas fibers under relaxing and contracting conditions. The first two inhibitors suppressed the contractile force almost completely at a 100 μM concentration, and a similar effect was obtained at 50 mM for BDM. However, still substantial changes were observed in the diffraction patterns upon calcium-activation of inhibited muscle fibers. (1) The 2nd actin layer-line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer-line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a saturating concentration, myosin heads leave the helix on the thick filaments and approach the thin filaments. Interestingly, the d1,0 spacing of the filament lattice remained unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.

An insight in the structure of a palygorskite from Palygorskaja: Some questions on the standard model

In this study we analyze in detail a palygorskite from Palygorskaja. This palygorskite is situated in the context of the existing studies using X-ray powder diffraction analysis. Moreover, a novel microdiffraction study on a small bunch of fibers shows highly structured 2D diffraction patterns that allow to decipher some information on the microstructure, thus overcoming the uncertainty usually originated by large samples containing mixtures and impurities. Structural data provided by Chisholm (1992) are used to simulate 1D and 2D powder and fiber diffraction patterns for palygorskite, which are compared with the experimental results. We performed simulations for powder and fiber diffraction and we centered our attention in the region of interest with d-spacings between 4.0 and 4.5Å. This palygorskite is consistent with a purely orthorhombic palygorskite, based on good agreement of data with simulations. The experimental results present some reflections not found in the simulations. These reflections are interpreted as corresponding to other hk1 planes of palygorskite. They do not match any reflection from the monoclinic structural model nor from probable impurities, reinforcing the interpretation of them being intrinsic to the structure of the mineral. Our findings suggest a revisal of the commonly accepted structure of palygorskite. They correspond to hk1 planes of palygorskite, and they do not match any reflection neither from the monoclinic structural model nor from possible impurities, and thus reinforcing the idea of them being intrinsic to the structure of the mineral, suggesting the necessity of a revisal of the commonly accepted structure of palygorskite.

Calculation of the cross-sectional shape of a fibril from equatorial scattering.

An alternate formulation of helical diffraction theory is used to generate cross-sectional shapes of fibrous structures from equatorial scattering. We demonstrate this approach with computationally generated scattering intensities and then apply it to scattering data from Tobacco Mosaic Virus (TMV) and in vitro assembled fibrils of Aβ40 peptides. Refining the cross-sectional shape of TMV from SAXS data collected on a 26mg/ml solution resulted in a circular shape with outer diameter of ∼180Å and inner diameter of ∼40Å consistent with the known structure of TMV. We also utilized this method to analyze the equatorial scattering from TMV collected by Don Caspar from a concentrated (24% ∼295mg/ml) gel of TMV as reported in his Ph.D. thesis in 1955. This data differs from the SAXS data in having a sharp interference peak at ∼250Å spacing, indicative of strong interparticle interactions in the gel. Analysis of this data required consideration of interatomic vectors as long as 2000Å and resulted in generation of images that were interpreted as representative of local organization of TMV particles in the sample. Peaks in the images were separated, on average by about 250Å with a density consistent with Caspar's original measurements. Analysis of SAXS data from Aβ fibrils resulted in a cross-sectional shape that could be interpreted in terms of structural models that have been constructed from ssNMR and cryoEM. These results demonstrate an unexpected use of the small-angle region of fiber diffraction patterns to derive fundamental structural properties of scattering objects.

Physical Properties of Plant Fibers (Sisal, Coir) and Its Composite Material with Tamarind Seed Gum as Low-Cost Housing Material

ABSTRACTIn this study, the physical properties of sisal and coir fibers have been described. Using manual extraction procedures, the sisal fibers were extracted from the sisal plant and coir fibers from the coconut palm. Thermal analysis by differential scanning calorimetry, structural morphology by scanning electron microscopy, and the degree of sharpness of the equatorial reflections of the X-ray fiber diffraction pattern were recorded for the untreated sisal and coir fibers. Tensile strength was determined for the single fiber and the results are correlated with the tensile strength of bundle fibers. Composite material has been prepared with this plant fiber as a filler and tamarind seed gum as a matrix material. The endosperms of roasted tamarind seeds were used for the preparation of tamarind seed gum solution. The different temperature condition maintained for roasting the seeds are 130°C, 160°C, and 180°C. The tensile strength of the prepared composite material is measured and it shows dependency of the roasting temperature condition of the tamarind seed. Scanning electron microscopy and water resistivity test were conducted, and the results were reported for the prepared composite material. Low-cost housing is made using this biodegradable sisal fiber--tamarind seed gum composite material.

An Obsevation of Native Skeletal Thin Filament Structure at High Calcium Condition by Electron Cryomicroscopy

It is hampering our understanding on the molecular mechanism of muscle contraction thin filament-linked modulation lack of electron cryomicryoscopy data supporting the steric model (Nature 368,64-67,1994). Recently well-oriented sols of flamentous (F)-actin formed by Ca2+-actin and length controlled by gelsolin give detailed X-ray fibre diffraction patterns led to the flat-actin conformation resolved in F-actin (Nature 457,441-550,2009). von der Eckon et al (Nature,519,114-119,2015) defined a three-dimensional structure of F-actin at a resolution of 3.7A and in synthetic complex with tropomyosin (no troponin complex) at a resolution of 6.5A using electron cryomicroscopy. Here is the first report about a well-defined structure of native skeletal thin filament at high calcium condition by electron cryomicroscopy with Iterative Helical Real-Space Reconstruction (IHRSR): tropomyosin locates C-state on actin, spreads over one of myosin subfragment-1 (S1) strongly binding sites between subdomain_I and_III of the actin (JMB,266,8-14,1997); one intra-strand contact with strong 3D-reconstructed density and more than one inter-strand contacts are resolved among neighboring actin monomers (n, n+1, n+2), mainly formed through actin hydrophobic plug 256-271 linked to various regions of the opposite actin subunits, different from those of S1 decorated F-actin and the published F-actin and F-actin-Tropomyosin complex, in the native skeletal thin filament suggesting actin conformation diversity—globular-actin conformation dominating in high calcium condition, the flat conformation coexisting, and the two identified actin conformations are dynamic in thin filaments during the modulation of skeletal muscle contraction.

Thin Filament Structure and the Steric Blocking Model.

By interacting with the troponin-tropomyosin complex on myofibrillar thin filaments, Ca2+ and myosin govern the regulatory switching processes influencing contractile activity of mammalian cardiac and skeletal muscles. A possible explanation of the roles played by Ca2+ and myosin emerged in the early 1970s when a compelling "steric model" began to gain traction as a likely mechanism accounting for muscle regulation. In its most simple form, the model holds that, under the control of Ca2+ binding to troponin and myosin binding to actin, tropomyosin strands running along thin filaments either block myosin-binding sites on actin when muscles are relaxed or move away from them when muscles are activated. Evidence for the steric model was initially based on interpretation of subtle changes observed in X-ray fiber diffraction patterns of intact skeletal muscle preparations. Over the past 25 years, electron microscopy coupled with three-dimensional reconstruction directly resolved thin filament organization under many experimental conditions and at increasingly higher resolution. At low-Ca2+, tropomyosin was shown to occupy a "blocked-state" position on the filament, and switched-on in a two-step process, involving first a movement of tropomyosin away from the majority of the myosin-binding site as Ca2+ binds to troponin and then a further movement to fully expose the site when small numbers of myosin heads bind to actin. In this contribution, basic information on Ca2+-regulation of muscle contraction is provided. A description is then given relating the voyage of discovery taken to arrive at the present understanding of the steric regulatory model.

Structure of the toxic core of α-synuclein from invisible crystals.

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

Alternative hydrogen bond models of cellulose II and IIII based on molecular force-fields and density functional theory

Alternative hydrogen-bond structures were found for cellulose II and IIII based on molecular dynamics simulations using four force fields and energy optimization based on density functional theory. All the modeling results were in support to the new hydrogen-bonding network. The revised structures of cellulose II and IIII differ with the fiber diffraction models mainly in the orientation of two hydroxyl groups, namely, OH2 and OH6 forming hydrogen-bond chains perpendicular to the cellulose molecule. In the alternative structures, the sense of hydrogen bond is inversed but little difference can be seen in hydrogen bond geometries. The preference of these alternative hydrogen bond structures comes from the local stabilization of hydroxyl groups with respect to the β carbon. On the other hand when simulated fiber diffraction patterns were compared with experimental ones, the current structure of cellulose II with higher energy and the alternative structure of cellulose IIII with lower energy were in better agreement.

X-Ray Diffraction Patterns of Oil Palm Empty Fruit Bunch Fibers with Varying Crystallinity

Crystallinity of oil palm fiber from empty fruit bunch (EFB) with and without tretaments was studied by analyzing the X-ray diffraction (XRD) pattern. In this paper, we focused on the effect of acid hydrolysis onto EFB on the crystallinity of the extracted cellulose. The reaction was carried out by soaking EFB in 1% (v/v) aqueous sulfuric acid (H2SO4) at different temperatures of 120, 130 and 140°C for 1 h. The XRD patterns significantly showed changes in the 2θ peaks before and after the treatment. These changes were described in term of polymorphs type present, reflection and allomorphs of the samples. XRD peak high and XRD deconvolution methods were used to calculate and compare the percentage of crystallinity of untreated EFB (UT-EFB) and acid hydrolyzed samples (AH-EFB). Based on the calculation, increment of about 1.3 times and 1.5 times were achieved by using WAXS and XRD deconvolution methods respectively. This is due to the removal of amorphous part contributed by lignin, hemicellulose and cellulose. Fourier Transform infrared (FTIR) spectra showed the presence of similar peaks in AH-EFB and commercial microcrystalline cellulose (C-MCC) at 1427, 1315, 895 and 1022 cm-1. The micrographic features showed the acid hydrolysis had successfully took place and separated the EFB microfibrils bundles.

Fibre Diffraction Analysis of Skin Offers a Very Early and Extremely Accurate Diagnostic Test for Prostate Cancer

Double blind analysis of a batch of thirty skin tissue samples from potential prostate cancer sufferers correctly identified all “control” patients, patients with high and low grade prostate cancers, the presence of benign prostate hyperplasia (BPH), perineural invasions, and the one lymphatic invasion. Identification was by analysis of fibre diffraction patterns interpreted using a schema developed from observations in nine previous studies. The method, schema, and specific experiment results are reported in this paper, with some implications then drawn.