RESUMO
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.
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The structure of the thin, actin-containing filament of muscle is both highly conserved across a broad range of muscle types and is now well understood. The structure of the thick, myosin-containing filaments of striated muscle are quite variable and remained comparatively unknown until recently, particularly in the arrangement of the myosin tails. John Squire played a major role not only in our understanding of thin filament structure and function but also in the structure of the thick filaments. Long before much was known about the structure and composition of muscle thick filaments, he proposed a general model for how myosin filaments were constructed. His role in our current understanding the structure of striated muscle thick filaments and the extent through which his predictions have held true is the topic of this review.
Assuntos
Miosinas , Sarcômeros , Miosinas/química , Músculo Esquelético , Citoesqueleto de ActinaRESUMO
Muscle is highly organized across multiple length scales. Consequently, small changes in the arrangement of myofilaments can influence macroscopic mechanical function. Two leg muscles of a cockroach have identical innervation, mass, twitch responses, length-tension curves and force-velocity relationships. However, during running, one muscle is dissipative (a 'brake'), while the other dissipates and produces significant positive mechanical work (bifunctional). Using time-resolved X-ray diffraction in intact, contracting muscle, we simultaneously measured the myofilament lattice spacing, packing structure and macroscopic force production of these muscles to test whether structural differences in the myofilament lattice might correspond to the muscles' different mechanical functions. While the packing patterns are the same, one muscle has 1â nm smaller lattice spacing at rest. Under isometric stimulation, the difference in lattice spacing disappeared, consistent with the two muscles' identical steady-state behavior. During periodic contractions, one muscle undergoes a 1â nm greater change in lattice spacing, which correlates with force. This is the first identified structural feature in the myofilament lattice of these two muscles that shares their whole-muscle dynamic differences and quasi-static similarities.
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Baratas , Miofibrilas , Citoesqueleto de Actina , Animais , Contração Muscular , Músculo Esquelético , SarcômerosRESUMO
Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Contração Isométrica/fisiologia , Músculo Estriado/fisiologia , Miosinas/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Simulação por Computador , Conectina/fisiologia , Conectina/ultraestrutura , Modelos Biológicos , Método de Monte Carlo , Músculo Estriado/diagnóstico por imagem , Miosinas/ultraestrutura , Rana catesbeiana/fisiologia , Sarcômeros/ultraestrutura , Espalhamento a Baixo Ângulo , Técnicas de Cultura de Tecidos , Difração de Raios XRESUMO
The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470â¯Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections zaâ¯â¼â¯74â¯Å and zbâ¯â¼â¯198â¯Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.
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Cabelo/química , Filamentos Intermediários/química , Queratinas Específicas do Cabelo/química , Porcos-Espinhos/metabolismo , Animais , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Modelos Químicos , Oxirredução , Difração de Raios XRESUMO
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 Aß40 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 â¼180Å and inner diameter of â¼40Å 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% â¼295mg/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 â¼250Å 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 Aß 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.
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Peptídeos beta-Amiloides/química , Modelos Teóricos , Vírus do Mosaico do Tabaco/química , Amiloide/química , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Silks from the Hymenoptera aculeata (bees, wasps, ants) contain ropes with four α-helical strands, rather than the more usual two strands found, for example, in α-keratin and myosin molecules. Extensive studies of the chemical structure of the silks have shown that each of the four chains in the molecule contains a central coiled-coil rod domain. However, little progress has been made in modeling the three-dimensional structure. X-ray diffraction data on honeybee silk (Apis mellifera), recorded by Rudall and coworkers, has been re-examined in detail and possible structures developed for the various types of filament seen in the silk glands, and for the packing arrangement in the spun fibers. The original X-ray data were re-collected by scanning figures in the original publications, de-screening and averaging perpendicular to the direction of interest, thereby reducing the graininess of the original images. Sufficient numbers of equatorial and meridional reflections were collected to define the axial projection of the base of the unit cell in fibers drawn from the contents of the silk glands, and to suggest that the axial period is different from that suggested by Rudall and coworkers. Models for two types of filament of increasing diameter are developed based on the node-internode packing scheme observed in protein crystals containing four-strand α-helical ropes. The central domains of the four component chains in the molecule are enclosed by N- and C-terminal domains with widely different lengths and compositions. The fibers thus have a composite filament-matrix texture, and possible locations for the matrix are discussed.
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Abelhas/metabolismo , Proteínas de Insetos/ultraestrutura , Seda/ultraestrutura , Vespas/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
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 ß-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 ß-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked ß-sheet fibrils nucleate the formation of the infectious ß-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious ß-solenoid fibrils) fibrillization. Several serial passages of stacked ß-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.
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Amiloide/química , Proteínas Fúngicas/química , Peptídeos/química , Príons/química , Amiloide/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Podospora/genética , Podospora/metabolismo , Príons/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Difração de Raios XRESUMO
This paper describes a method for recording X-ray diffraction patterns from a small amount of fibrous protein materials while being oriented by using a micro shear-flow cell. This cell consists of two concentrically arranged glass tubes. The inner tube is stationary, while the outer one rotates at a high speed. The gap between the two tubes is about 100 µm, into which the suspension of fibrous protein materials is injected. By using synchrotron-radiation X-ray microbeams (diameter, 10 µm), clear diffraction images from oriented protein materials can be recorded. The required volume of the sample is only about 10 µl. This method can also be combined with the laser-flash photolysis of caged compounds. Examples of application of this method to the flagella of a green alga Chlamydomonas, and sperm of a tunicate Ciona are presented.
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This article presents an upgrade of the D+ software [Ginsburg et al. (2019 â¸). J. Appl. Cryst. 52, 219-242], expanding its hierarchical solution X-ray scattering modeling capabilities for fiber diffraction and single crystallographic orientations. This upgrade was carried out using the reciprocal grid algorithm [Ginsburg et al. (2016 â¸). J. Chem. Inf. Model. 56, 1518-1527], providing D+ its computational strength. Furthermore, the extensive modifications made to the Python API of D+ are described, broadening the X-ray analysis performed with D+ to account for the effects of the instrument-resolution function and polydispersity. In addition, structure-factor and radial-distribution-function modules were added, taking into account the effects of thermal fluctuations and intermolecular interactions. Finally, numerical examples demonstrate the usage and potential of the added features.
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Polysaccharides are the most abundant class of biopolymers, holding an important place in biological systems and sustainable material development. Their spatial organization and intra- and intermolecular interactions are thus of great interest. However, conventional single crystal crystallography is not applicable since polysaccharides crystallize only into tiny crystals. Several crystallographic methods have been developed to extract atomic-resolution structural information from polysaccharide crystals. Small-probe single crystal diffractometry, high-resolution fiber diffraction and powder diffraction combined with molecular modeling brought new insights from various types of polysaccharide crystals, and led to many high-resolution crystal structures over the past two decades. Current challenges lie in the analysis of disorder and defects by further integrating molecular modeling methods for low-resolution diffraction data.
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Polissacarídeos , Cristalografia , Cristalografia por Raios X , Modelos MolecularesRESUMO
X-ray/neutron fiber diffraction and small-angle X-ray/neutron scattering are widely used to investigate the molecular structure of fibrous proteins, including amyloid fibrils. However, there is sometimes confusion between these two techniques despite the fact that sample conditions and the content of the information obtained are not the same. In this brief chapter, we present the differences in sample conditions between these two methods, and their effects on experimentally obtained diffraction or scattering patterns, emphasizing the degree of disorder in the samples.
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Amiloide , Nêutrons , Amiloide/química , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Over the last decade, the structural refinement of cellulose allomorphs and their analogs has been advanced using high-resolution fiber diffraction. This also includes structures of crystals complexed with small molecules, which can inherently involve disorders. Computational methods, including density functional theory, in combination with molecular modeling are leading to improved structural analyses. Spectroscopic techniques such as vibrational spectroscopy give quantitative and robust data directly related to structural insights on cellulose. These data will benefit from improved molecular modeling's capacity for interpretation and will also serve as a gauge to measure the capacity of molecular modeling as an aid in structural determinations. Improvement in the capacity to directly simulate experimental data such as that from scattering, diffraction, and spectra will be the key for further integration of modeling and experimental approaches.
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Celulose , Vibração , Modelos Moleculares , Análise EspectralRESUMO
We determined the molecular and packing structure of a chitosan-ZnCl2 complex by X-ray diffraction and linked-atom least-squares. Eight D-glucosamine residues-composed of four chitosan chains with two-fold helical symmetry, and four ZnCl2 molecules-were packed in a rectangular unit cell with dimensions a = 1.1677 nm, b = 1.7991 nm, and c = 1.0307 nm (where c is the fiber axis). We performed exhaustive structure searches by examining all of the possible chain packing modes. We also comprehensively searched the positions and spatial orientations of the ZnCl2 molecules. Chitosan chains of antiparallel polarity formed zigzag-shaped chain sheets, where N2···O6, N2···N2, and O6···O6 intermolecular hydrogen bonds connected the neighboring chains. We further refined the packing positions of the ZnCl2 molecules by theoretical calculations of the crystal models, which suggested a possible coordination scheme of Zn(II) with an O6 atom.
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The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7-10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.
Assuntos
Epiderme/química , Filamentos Intermediários/química , beta-Queratinas/química , Animais , Evolução Biológica , Aves , Domínios Proteicos , RépteisRESUMO
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.
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Ab initio reconstruction methods have revolutionized the capabilities of small-angle X-ray scattering (SAXS), allowing the data-driven discovery of previously unknown molecular conformations, exploiting optimization heuristics and assumptions behind the composition of globular molecules. While these methods have been successful for the analysis of small particles, their impact on fibrillar assemblies has been more limited. The micrometre-range size of these assemblies and the complex interaction of their periodicities in their scattering profiles indicate that the discovery of fibril structures from SAXS measurements requires novel approaches beyond extending existing tools for molecular discovery. In this work, it is proposed to use SAXS measurements, together with diffraction theory, to infer the electron distribution of the average cross-section of a fiber. This cross-section is modeled as a discrete electron density with continuous support, allowing representations beyond binary distributions. Additional constraints, such as non-negativity or smoothness/connectedness, can also be added to the framework. The proposed approach is tested using simulated SAXS data from amyloid ß fibril models and using measured data of Tobacco mosaic virus from SAXS experiments, recovering the geometry and density of the cross-sections in all cases. The approach is further tested by analyzing SAXS data from different amyloid ß fibril assemblies, with results that are in agreement with previously proposed models from cryo-EM measurements. The limitations of the proposed method, together with an analysis of the robustness of the method and the combination with different experimental sources, are also discussed.
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Amiloide/química , Espalhamento a Baixo Ângulo , Vírus do Mosaico do Tabaco/química , Difração de Raios X/métodos , Algoritmos , Microscopia Crioeletrônica , Modelos Moleculares , SoftwareRESUMO
Wandering spiders climb vertically and walk upside-down on rough and smooth surfaces using a nanostructured attachment system on their feet. The spiders are assumed to adhere by intermolecular van der Waals forces between the adhesive structures and the substrate. The adhesive elements are arranged highly ordered on the hierarchically structured attachment hair (setae). While walking, it has been suggested that the spiders apply a shear force on their legs to increase friction. However, the detailed mechanical behavior of the hair's structures during attachment and detachment remains unknown. Here, gradients of the mechanical properties of the attachment hair on different length scales that have evolved to support attachment, stabilize adhesion in contact, and withstand high stress at detachment, examined by in situ experiments, are shown. Shearing helps to self-align the adhesive elements with the substrate. The study is anticipated to contribute to the development of optimized artificial dry adhesives.
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Cabelo , Fenômenos Mecânicos , Aranhas/anatomia & histologia , Adesividade , Animais , Fenômenos Biomecânicos , Estresse MecânicoRESUMO
Many proteins and peptides are able to self-assemble in solution in vitro and in vivo to form amyloid-like fibrils. These fibrils share common structural characteristics. In order for a fibril to be characterized as amyloid, it is expected to fit certain criteria including the composition of cross-ß. Here we describe how the formation of amyloid fibrils can be characterized in vitro using a variety of methods including circular dichroism and intrinsic tyrosine/tryptophan fluoresence to follow conformational changes; Thioflavin and/or ThS assembly to monitor nucleation and growth; transmission electron microscopy to visualize fibrillar morphology and X-ray fiber diffraction to examine cross-ß structure.
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Amiloide/química , Proteínas Amiloidogênicas/química , Modelos Moleculares , Conformação Proteica , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Proteínas Amiloidogênicas/metabolismo , Benzotiazóis/química , Benzotiazóis/metabolismo , Dicroísmo Circular , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/metabolismo , Relação Quantitativa Estrutura-Atividade , Difração de Raios XRESUMO
We present a detailed study on the self-assembly and cytotoxicity of arginine-rich fragments with general form [RF]n (n=1-5). These highly simplified sequences, containing only two l-amino acids, provide suitable models for exploring both structure and cytotoxicity features of arginine-based oligopeptides. The organization of the sequences is revealed over a range of length scales, from the nanometer range down to the level of molecular packing, and their cytotoxicity toward C6 rat glioma and RAW264.7 macrophage cell lines is investigated. We found that the polymorphism is dependent on peptide length, with a progressive increase in crystalline ordering upon increasing the number of [RF] pairs along the backbone. A dependence on length was also found for other observables, including critical aggregation concentrations, formation of chiral assemblies and half maximum inhibitory concentrations (IC50). Whereas shorter peptides self-assemble into fractal-like aggregates, clear fibrillogenic capabilities are identified for longer sequences with octameric and decameric chains exhibiting crystalline phases organized into cross-ß structures. Cell viability assays revealed dose-dependent cytotoxicity profiles with very similar behavior for both glioma and macrophage cell lines, which has been interpreted as evidence for a nonspecific mechanism involved in toxicity. We propose that structural organization of [RF]n peptides plays a paramount role regarding toxicity due to strong increase of local charge density induced by self-assemblies rich in cationic groups when interacting with cell membranes.