The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion.

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

A consensus model for molecular packing of type I collagen.

In this review, recent results from X-ray diffraction studies of tendon are used to develop an understanding of the molecular packing of type I collagen in tendon fibrils. These cover the definition of the unit cell as triclinic, the lateral architecture of molecular packing in a fibril and the molecular packing topology of a structure that gives good agreement with X-ray diffraction data. The proposed model is a 1D staggered left handed microfibril; the molecular orientation of the telopeptides indicates that there are interconnections between microfibrils that may explain the difficulty in isolating individual microfibrillar structures. This is the first structure that defines the absolute molecular packing of molecular segments based on X-ray diffraction data. These results are discussed in the light of direct and indirect evidence relating to molecular packing such as mineralization, natural crosslink position, and biomechanical evidence. The ability of the proposed structure to fulfill many of the structural and biochemical criteria point towards the structure providing a basis for a consensus model of collagen packing.

Molecular packing of type I collagen in tendon.

X-ray diffraction of rat tail tendon shows that type I collagen fibrils contain regions of three-dimensional crystalline arrays; where molecular packing is speculated to be by a staggered sheet or microfibril arrangement. The X-ray diffraction pattern also contains a significant amount of diffuse scatter indicative of static and thermal disorder in fibrils. Removal of the diffuse scatter from the equatorial region of X-ray diffraction patterns obtained using synchrotron radiation allowed the Bragg intensities to be viewed on a flat background. Indexing of Bragg peak intensity on the 10, -10, 0 -1, 01, -11 and 1-1 row-lines of the triclinic unit cell have been used here to test possible sheet and microfibril packing arrangements. The relative translation of molecular segments in the gap and overlap regions as well as the telopeptide orientation have been investigated. A global search through combinations of molecular packing and molecular translation revealed that the sheet-type conformations cannot account for the observed low-angle off-meridional Bragg peak intensity distribution. A superior fit is obtained with D-staggered left-handed microfibril structures. The orientation of the telopeptides may indicate that there are interconnections between microfibrils that may explain the difficulty in isolating individual microfibrillar structures.

Calibration and application of an X-ray image intensifier/ charge-coupled device detector for monochromatic macromolecular crystallography.

Charge-coupled device (CCD)-based X-ray detectors allow data to be collected much more quickly (approximately 10 times) than with current on-line imaging-plate systems. At the ESRF, X-ray image intensifier/CCD detector systems have been developed. These have great potential as fast read-out detectors for macromolecular and other forms of crystallography. They are relatively large sensitive X-ray detectors but have two inherent weaknesses: convex detection surfaces leading to spatial distortion and non-uniformity of intensity response, and susceptibility to small changes in magnetic fields. A large improvement has been made to the accuracy obtained by non-uniformity of response calibration and correction, using fluorescence from doped lithium borate glasses. Monochromatic macromolecular crystallography demonstration experiments with external user groups have shown that high-quality results may be obtained under real experimental conditions.

A Novel Technique for Accurate Intensity Calibration of Area X-ray Detectors at Almost Arbitrary Energy.

A novel intensity uniformity calibration method for area X-ray detectors is described. In diffraction experiments, amorphous lithium glass plates, containing doping elements chosen for their K edges just below the energy of the main beam, replace the crystallographic samples for the calibration measurement. The fluorescent emission excited by the X-ray beam is almost isotropic. It has exactly the same geometry as the diffracted radiation, and can be obtained at the same wavelength by proper selection of the element and excitation energy. A sample 2theta scan allows the emission distribution as a function of angle to be characterized with an accuracy of a fraction of a percent. This allows a flat-field correction of similar accuracy. The quality of crystallographic data collected with an X-ray image intensifier/CCD detector was significantly improved by flat-field correction using an Sr-doped lithium tetraborate glass. This technique can be applied to X-ray energies from 5 to 50 KeV; the calibration sample is small, stable and easily handled.

Human IgG subclass responses and subclass restriction to Schistosoma mansoni egg antigens.

In areas endemic for schistosomiasis, there is great heterogeneity in antibody isotype responses to parasite antigens amongst infected individuals. At the population level, the isotype composition of antibody responses undergoes dynamic changes which are associated with the age of infected individuals. Here we examine the IgG subclass responses to Schistosoma mansoni eggs (soluble egg antigens; SEA) of infected individuals by immunoblot and ELISA. By controlled treatment of SEA-coated ELISA plates and immunoblot nitrocellular strips with sodium periodate, in order to oxidize terminal carbohydrate residues selectively, we were able to relate individuals subjects' isotype responses to the different antigens that they responded to, and to the presence of putative carbohydrate and peptide epitopes on those antigens. IgG2 responses were restricted strictly to sodium periodate-sensitive carbohydrate epitopes and antigens of relatively high molecular weight. These antigens were not usually recognized by other isotypes and, therefore, they were only recognized by individuals who had high levels of IgG2. IgG1 and IgG3 responses were directed against both carbohydrate and peptide epitopes, whereas IgG4 responses were restricted to periodate-resistant epitopes. This suggests that the fall in IgG2 responses, and reciprocal rise in IgG4 antibodies, seen in young children as their intensities of schistosome infection increase, is not the result of isotype switching, and that, if these two subclasses are involved in blocking immunity to schistosomiasis, they are operating independently.