CLEARER: a new tool for the analysis of X-ray fibre diffraction patterns and diffraction simulation from atomic structural models

Fibre diffraction can provide structural information about polymers and biopolymers that is unobtainable using other methods. This method has been used to elucidate the structures of many polymers, biopolymers and protein assemblies. Extracting structural information from fibre diffraction patterns is a major challenge. A computer program called CLEARER has been developed that aids the detailed analysis of polycrystalline fibre diffraction patterns. It offers an easy-to-use interface that enables diffraction data processing, analysis and simulation of diffraction patterns. It is likely to be applicable to structural determination for a wide range of polymeric fibrous materials. CLEARER simplifies and speeds up the data analysis process and helps to utilize all of the structural information present in the analysed X-ray and electron diffraction patterns

X-Ray Diffraction Studies of Amyloid Structure

Elucidation of the underlying core structure of amyloid fibrils is essential for understanding the mechanism by which amyloid fibrils are formed and deposited. Conventional methods of X-ray crystallography and NMR cannot be used, since the fibers are insoluble and heterogeneous. X-ray fiber diffraction is one method that has been successfully used to examine the structure of these insoluble fibers. The procedure involves the formation of suitable, ordered amyloid fibrils and characterization (by electron microscopy), partial alignment of fibers, X-ray data collection, data analysis, and finally, model building

The Structure of Amyloid

The local or systemic deposition of insoluble amyloid fibrils is characteristic of the pathogenesis of the heterogeneous group of diseases known as the amyloidoses. Normally soluble, innocuous proteins undergo a change in conformation and self assemble into insoluble, potentially toxic, amyloid fibrils. Electron microscopy shows amyloid fibrils to be straight, unbranching structures, 70 to 120 Å in diameter and of indeterminate length. The potential for amyloidogenesis may be a near universal property of protein. Knowledge of the structure of these fibrils is a crucial element in the development of an understanding of their stability and assembly. With this information, the rational design of drugs to prevent amyloidogenesis and promote disassembly might be enabled. Furthermore, it may grant some insight into the generality of protein folding. Single crystal X-ray crystallography and solution NMR are not possible due to the fibrillar inability to crystallise and to intrinsic insolubility. X-ray and recently electron fibre diffraction have proved to be of great value in the elucidation of the structure of amyloid. This review discusses the advances made and how fibre diffraction is used in conjunction with other structural technique

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 Å apart. Electron diffraction also revealed the characteristic 4.7 Å meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 Å that has been previously visualised from a single Aß fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded ß-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils

Diffraction to study protein and peptide assemblies

Proteins and peptides are able to self-assemble in vivo and in vitro. In vitro, this ability can be exploited to make bionanomaterials with many potential uses. Peptides are capable of forming a wide range of structures including fibres, tubules and scaffolds. In vivo, proteins assemble to form cellular fibrous proteins, as well as being involved in protein misfolding in disease. Recent advances using X-ray diffraction have highlighted the internal structure of self-assembled proteins and peptides, showing packing of side chain residues and have enabled a deeper understanding of mechanisms of assembly

Molecular basis for amyloid fibril formation and stability

The molecular structure of the amyloid fibril has remained elusive because of the difficulty of growing well diffracting crystals. By using a sequence-designed polypeptide, we have produced crystals of an amyloid fiber. These crystals diffract to high resolution (1 Å) by electron and x-ray diffraction, enabling us to determine a detailed structure for amyloid. The structure reveals that the polypeptides form fibrous crystals composed of antiparallel ß-sheets in a cross-ß arrangement, characteristic of all amyloid fibers, and allows us to determine the side-chain packing within an amyloid fiber. The antiparallel ß-sheets are zipped together by means of p-bonding between adjacent phenylalanine rings and salt-bridges between charge pairs (glutamic acid¿lysine), thus controlling and stabilizing the structure. These interactions are likely to be important in the formation and stability of other amyloid fibrils

The Common Architecture of Cross-beta Amyloid.

Amyloid fibril deposition is central to the pathology of more than 30 unrelated diseases including Alzheimer's disease and Type 2 diabetes. It is generally accepted that amyloid fibrils share common structural features despite each disease being characterised by the deposition of an unrelated protein or peptide. The structure of amyloid fibrils has been studied using X-ray fibre diffraction and crystallography, solid-state NMR and electron paramagnetic resonance, and many different, sometimes opposing, models have been suggested. Many of these models are based on the original interpretation of the cross-beta diffraction pattern for cross-beta silk in which beta-strands run perpendicular to the fibre axis, although alternative models include p-helices and natively structured proteins. Here, we have analysed opposing model structures and examined the necessary structural elements within the amyloid core structure, as well as producing idealised models to test the limits of the core conformation. Our work supports the view that amyloid fibrils share a number of common structural features, resulting in characteristic diffraction patterns. This pattern may be satisfied by structures in which the strands align close to perpendicular to the fibre axis and are regularly arranged to form beta-sheet ribbons. Furthermore, the fibril structure contains several beta-sheets that associate via side-chain packing to form the final protofilament structure

Hydrogels formed from Fmoc amino acids

A number of Fmoc amino acids can be effective low molecular weight hydrogelators. The type of gel formed depends on the amino acid used and, in the case of FmocF, the final pH of the system. The single crystal structure of two of the gelators (FmocF and FmocY) have been determined and the data compared to the fibre X-ray diffraction data. FmocF, which forms metastable gels, crystallises easily and the data for the fibre phase and crystal phase are relatively similar. For FmocF, the fibre axis in b is consistent with the hydrogen bonding repeat distances and the diffraction pattern calculated from the single crystal structure is a good match with the experimental fibre X-ray diffraction data. On the other hand, there are significant differences between the crystalline phase determined and the fibre phase for FmocY. The packing of FmocY within the crystal structure is created by interactions between the planar Fmoc groups, whilst it is clear that hydrogen bonding drives the self-assembly into fibrillar structures within the gels. This shows that understanding the packing in gel phase by analogy to isolated crystal structures has the potential to lead to erroneous conclusions