X-ray 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.

Interpreting equatorial X-ray diffraction patterns from striated muscle with multiscale modeling.

Small-angle X-ray diffraction is the tool of choice to obtain nm-scale structural information from striated muscle under near-physiological conditions. A critical barrier to progress has been the lack of generally applicable computational tools for modeling these X-ray diffraction patterns, limiting our ability to extract all the structural information potentially available. We addressed this need by extending the spatially explicit simulation platform MUSICO to predict equatorial fiber X-ray diffraction patterns from skeletal muscle. Electron densities for thin filaments with bound crossbridges were calculated using PDB structures of myosin S1, actin, tropomyosin, troponin and analogous nebulin structure derived from tropomyosin PDB. Thick filament backbone densities were constructed based on the Squire 1973 model incorporating “ribbon motifs” from a high-resolution cryo-EM structure of the Lethocerus thick filament. Titin and myosin S2 regions were constructed as cylindrical rings of density shells. Myosin heads in an ordered “parked state” were positioned in a shell close to the thick filament backbone with any disordered myosin heads distributed in a shell extending out to actin. X-ray diffraction patterns were calculated from sub-system motifs consisting of 1 thick filament and two thin filaments by evaluating the Fourier transform of this object at the reciprocal hexagonal lattice positions. Intensities were averaged over the 500 thick filaments and 1000 thin filaments used in simulation. Using this approach, we found that we could recapitulate 7 independent experimental intensities (out to the 4,0 reflection) from relaxed rat soleus and EDL muscle by varying only the proportion of heads in the parked state and a temperature factor type disorder term. Fits to contracting data could be achieved by adjusting only the temperature factor term and the number of force-producing crossbridges predicted by MUSICO to match the force levels observed experimentally.

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.

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.

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.

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.

Supramolecular structure of highly oriented wet-spun polyacrylonitrile fibers used in the preparation of high-performance carbon fibers

Highly oriented polyacrylonitrile (PAN) fibers, which are used in the preparation of high-performance carbon fibers, were prepared via a wet spinning process. The supramolecular structure—i.e., the degree of crystallinity, crystal size, and crystallite orientation—of the PAN fibers was characterized by X-ray diffraction (XRD) using fiber and powder techniques. Two equatorial diffraction peaks with 2θ ∼ 17° and 29.5° were observed in the fiber XRD patterns, which confirmed that the crystalline structure of the PAN fibers was pseudohexagonal with only two-dimensional order in the transverse direction. The powder XRD patterns were resolved into three constituent Lorentz peaks to determine the relative degree of crystallinity. In order to increase the reproducibility of the value for the crystallinity of the fibers, the Gupta–Singhal method was modified by assuming that the degree of crystallinity could be determined from the area under peak 1 (with 2θ ∼ 17°), rather than the combined area of peaks 1 and 3. The evolution of the supramolecular structure of the PAN fibers during the spinning process was also investigated. Results indicated that drying and steam stretching play important roles in the formation and growth of the crystalline structure of the PAN fibers, while the orientation of the structure was only strongly related to the degree of stretching. The effects of the supramolecular structure on the thermal properties and mechanical properties of the PAN fibers were also studied. The perfection of the supramolecular structure influenced the feasibility of cyclization reactions within the PAN fibers and the thermal decomposition of those fibers. The mechanical properties of the PAN fibers were significantly enhanced by increasing the perfection of the supramolecular structure.

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.

X-Ray Diffraction Pattern of Non-Uniformly Stretched Actin Filament

Helical structures within living cells are exposed to mechanical forces and are consequently deformed. However, interpretations of X-ray fiber diffraction patterns of such structures assume Fourier transform of helices with fixed periodicities. Huxley et al. and Wakabayashi et al. (Biophys. J. 67: 2422-2435 and 2411-2421, 1994) calculated the extensibility of actin filaments in contracting muscle fibers from the X-ray diffraction patterns using mean values of strain. Single value of strain corresponds to symmetric, narrow meridional peak profile, while, non-uniform deformation of actin filament provides profile similar to the observations, noticeably changing elastic constant for actin filament. Due to discrete, stochastic attachments of myosin heads to actin filaments the strain along the filament increases stepwise from the free end of the actin filament to the Z-line. We have developed a methodology for predicting X-ray diffraction patterns with stepwise increases of strain along actin filament. Using PDB data for the crystal structures of G-actin and rules for constructing actin fibers we reconstructed the geometry of actual deformed fibers. Fiber deformations are determined by Monte Carlo calculations using the computational platform, MUSICO. Predicted X-ray diffraction patterns show smeared layer lines, caused by different pitch of helices, in contrast to distinct layer lines originating from constant pitch of undeformed helices. Calculated meridional X-ray diffraction peak profiles from deformed helices are skewed and closely resemble the observed profiles by Huxley et al. and Wakabayashi et al. The proposed methodology for analyzing deformed helices provides realistic intensity profiles and spacing values of molecular structures in inverse space. This enables the extraction of much more information concerning molecular structure from the X-ray diffraction data from any helical structures in living cells.Supported by: NIH 9 P41 GM103622, R01 AR048776 and R01 DC 011528.

Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region.

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

The Structure of Cross‐β Tapes and Tubes Formed by an Octapeptide, αSβ1

Elaborate morphology: The αSβ1 peptide, a fragment of α-synuclein, assembles into flat tapes consisting of a peptide bilayer, which can be modeled based on the cross-β structure found in amyloid proteins. The tapes are stabilized by hydrogen bonding, whilst the amphiphilic nature of the peptide results in the thin bilayer structure. To further stabilize the structure, these tapes may twist to form helical tapes, which subsequently close into nanotubes.

Determination of Ratio of Diamagnetic Anisotropy of a Biaxial Crystal by X-ray Diffraction Measurement

The ratio of diamagnetic anisotropy of a biaxial crystal (cellobiose: monoclinic, space group P21) was determined using two X-ray fiber diffraction patterns that were obtained from its microcrystalline powder oriented in static and rotating magnetic fields. We first determined the directions of the magnetic axes with respect to the crystallographic axes. We found that χ3 is parallel to the b*-axis, and the χ1-axis forms angles of 67.9 and 22.8° with respect to the a*- and c*-axes, respectively. Using the analysis method proposed recently, azimuthal half widths of the X-ray diffraction spots for (hkl) planes of the oriented samples were plotted as a function of sin 2Φ, where Φ is an angle that characterizes the direction of the reciprocal vector of the (hkl) plane in the χ1χ2χ3 coordinates, with χ1, χ2, and χ3 being the principal axes of the magnetic susceptibility tensor. The half width linearly depended on sin 2Φ as predicted by the proposed method. From the values of the slope and intercept of the plot, the ratio of the diamagnetic anisotropy, rχ=(χ2-χ3)/(χ1-χ2) was determined, where χ1>χ2>χ3. We estimated that rχ=1.4–1.7.

Trivalent Iron Induced Gelation in Lambda-Carrageenan.

This communication reports gelation of lambda-carrageenan, for the first time, in the presence of trivalent iron ions. Kappa-, iota- and lambda-carrageenans are sulfated polysaccharides used extensively in food, pharmaceutical and medical applications. Kappa- and iota-carrageenans show gelation in the presence of mono- and di-valent ions, but lambda-carrageenan yields only viscous solutions. Our results show that gelation in lambda-carrageenan indeed is possible, but with trivalent ions. X-ray fiber diffraction patterns of iron (III)-lambda-carrageenan are characteristic of highly oriented and polycrystalline fibers containing well resolved Bragg reflections. The elastic modulus (G') of the product is far greater than the loss modulus (G") indicating the thermal stability of lambda-carrageenan in the presence of iron (III) ions. This novel finding has potential to expand lambda-carrageenan's current utility beyond a viscosifying agent.

Variability in sepiolite: Diffraction studies

Twenty sepiolites of known composition from different origins were analyzed to quantify the variability in structural parameters and its possible relationships with composition and morphology. Morphology SEM analyses led to classify the sepiolites into several groups, beyond the two simple macroscopic or clay groups. X-ray powder diffraction with synchrotron light was used to discuss the variability of the a and b cell parameters with the nature of the cations and occupancy of the octahedral shell. Rietveld refinement using the ideal sepiolite model is performed on sepiolites at two temperatures: 225 °C (for zeolitically dehydrated sepiolite) and 25 °C (for hydrated ambient sepiolite). The latter permitted to locate ca. six molecules of the zeolitic H2O within the tunnels. A few samples were selected to evaluate the feasibility and potential of single-crystal diffraction methods: X-ray microdiffraction and electron diffraction. The macroscopic sepiolites gave well-structured and rich X-ray fiber diffraction patterns, in excellent agreement with ab initio simulations. High-quality single-crystal electron diffraction patterns for three axis zones are indexed and compared with simulations. The experimental and modeling results for X-ray microdiffraction and electron diffraction open a new path for quantitative crystallography on sepiolite and other fibrous clays from the sepiolite-palygorskite group.

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR.

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.

Structural Collagen Alterations in Macular Corneal Dystrophy Occur Mainly in the Posterior Stroma

Purpose: Collagen fibrils in the corneal stroma in macular corneal dystrophy, on average, are more closely spaced than in the normal cornea. This study was conducted to investigate if this occurs uniformly across the stroma or is more prevalent at certain stromal depths.Methods: Microbeam synchrotron X-ray fiber diffraction patterns were obtained in 25 μm steps across the whole thickness of a thin strip of a macular corneal dystrophy cornea obtained at keratoplasty. Data were analyzed for mean collagen interfibrillar spacing at all positions. Serum was analyzed immunochemically to determine immunophenotype, and transmission electron microscopy was carried out to visualize stromal ultrastructure.Results: Keratan sulphate was not detectable in blood serum, classifying the disease as macular corneal dystrophy type I. Collagen interfibrillar spacing dropped linearly with stromal depth from the anterior to posterior cornea, measuring 5–10% less in the posterior 100 µm of the MCD stroma compared to the anterior 100 µm (p < 0.001). Isolated pockets of collagen fibrils with unusually large diameters were identified in the deep stroma.Conclusions: Collagen fibril spacing is reduced and large-diameter collagen fibrils are seen in macular corneal dystrophy type I, with the deep stroma affected more. We speculate that the ultrastructural abnormalities are more prevalent in the posterior stroma because the structural influence of sulphated keratan sulphate glycosaminoglycans/proteoglycans is high in this region of the cornea.