IUCrData launches Raw Data Letters.

A new category of articles - Raw Data Letters - is introduced to IUCrData.

Rigid-body motion is the main source of diffuse scattering in protein crystallography.

The origin of diffuse X-ray scattering from protein crystals has been the subject of debate over the past three decades regarding whether it arises from correlated atomic motions within the molecule or from rigid-body disorder. Here, a supercell approach to modelling diffuse scattering is presented that uses ensembles of molecular models representing rigid-body motions as well as internal motions as obtained from ensemble refinement. This approach allows oversampling of Miller indices and comparison with equally oversampled diffuse data, thus allowing the maximum information to be extracted from experiments. It is found that most of the diffuse scattering comes from correlated motions within the unit cell, with only a minor contribution from longer-range correlated displacements. Rigid-body motions, and in particular rigid-body translations, make by far the most dominant contribution to the diffuse scattering, and internal motions give only a modest addition. This suggests that modelling biologically relevant protein dynamics from diffuse scattering may present an even larger challenge than was thought.

The structure of the FnI-EGF-like tandem domain of coagulation factor XII solved using SIRAS.

Coagulation factor XII (FXII) is a key protein in the intrinsic coagulation and kallikrein-kinin pathways. It has been found that negative surfaces and amyloids, such as Aß fibrils, can activate FXII. Additionally, it has been suggested that FXII simulates cells and that it plays an important role in thrombosis. To date, no structural data on FXII have been deposited, which makes it difficult to support any hypothesis on the mechanism of FXII function. The crystal structure of the FnI-EGF-like tandem domain of FXII presented here was solved using experimental phases. To determine the phases, a SIRAS approach was used with a native and a holmium chloride-soaked data set. The holmium cluster was coordinated by the C-terminal tails of two symmetry-related molecules. Another observation was that the FnI domain was much more ordered than the EGF-like domain owing to crystal packing. Furthermore, the structure shows the same domain orientation as the homologous FnI-EGF-like tandem domain of tPA. The plausibility of several proposed interactions of these domains of FXII is discussed. Based on this FXII FnI-EGF-like structure, it could be possible that FXII binding to amyloid and negatively charged surfaces is mediated via this part of FXII.

Microspheres with ultrahigh holmium content for radioablation of malignancies.

PURPOSE: The aim of this study was to develop microspheres with an ultra high holmium content which can be neutron activated for radioablation of malignancies. These microspheres are proposed to be delivered selectively through either intratumoral injections into solid tumors or administered via an intravascularly placed catheter. METHODS: Microspheres were prepared by solvent evaporation, using holmium acetylacetonate (HoAcAc) crystals as the sole ingredient. Microspheres were characterized using light and scanning electron microscopy, coulter counter, titrimetry, infrared and Raman spectroscopy, differential scanning calorimetry, X-ray powder diffraction, magnetic resonance imaging (MRI), and X-ray computed tomography (CT). RESULTS: Microspheres, thus prepared displayed a smooth surface. The holmium content of the HoAcAc microspheres (44% (w/w)) was higher than the holmium content of the starting material, HoAcAc crystals (33% (w/w)). This was attributed to the loss of acetylacetonate from the HoAcAc complex, during rearrangement of acetylacetonate around the holmium ion. The increase of the holmium content allows for the detection of (sub)microgram amounts of microspheres using MRI and CT. CONCLUSIONS: HoAcAc microspheres with an ultra-high holmium content were prepared. These microspheres are suitable for radioablation of tumors by intratumoral injections or treatment of liver tumors through transcatheter administration.

Influence of neutron irradiation on holmium acetylacetonate loaded poly(L-lactic acid) microspheres.

Holmium-loaded microspheres are useful systems in radio-embolization therapy of liver metastases. For administration to a patient, the holmium-loaded microspheres have to be irradiated in a nuclear reactor to become radioactive. In this paper. the influence of neutron irradiation on poly(L-lactic acid) (PLLA) microspheres and films, with or without holmium acetylacetonate (HoAcAc), is investigated, in particular using differential scanning calorimetry (MDSC), scanning electron microscopy, gel permeation chromatography (GPC), infrared spectroscopy, and X-ray diffraction. After irradiation of the microspheres, only minor surface changes were seen using scanning electron microscopy, and the holmium complex remained immobilized in the polymer matrix as reflected by a relatively small release of this complex. GPC and MDSC measurements showed a decrease in molecular weight and crystallinity of the PLLA, respectively, which can be ascribed to radiation induced chain scission. Irradiation of the HoAcAc loaded PLLA matrices resulted in evaporation of the non-coordinated and one coordinated water molecule of the HoAcAc complex, as evidenced by MDSC and X-ray diffraction analysis. Infrared spectroscopy indicated that some degradation of the acetylacetonate anion occurred after irradiation. Although some radiation induced damage of both the PLLA matrix and the embedded HoAcAc-complex occurs, the microspheres retain their favourable properties (no marginal release of Ho, preservation of the microsphere size), which make these systems interesting candidates for the treatment of tumours by radio-embolization.

Characterization of poly(L-lactic acid) microspheres loaded with holmium acetylacetonate.

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.

The crystal and molecular structures of cellulose I and II.

The paper describes molecular dynamics (MD) simulations on the crystal structures of the Ibeta and II phases of cellulose. Structural proposals for each of these were made in the 1970s on the basis of X-ray diffraction data. However, due to the limited resolution of these data some controversies remained and details on hydrogen bonding could not be directly obtained. In contrast to structure factor amplitudes in X-ray diffraction, energies, as obtained from MD simulations, are very sensitive to the positions of the hydroxyl hydrogen atoms. Therefore the latter technique is very suitable for obtaining such structural details. MD simulations of the Ibeta phase clearly shows preference for one of the two possible models in which the chains are packed in a parallel orientation. Only the parallel-down mode (in the definition of Gardner and Blackwell (1974) J Biopolym 13: 1975-2001) presents a stable structure. The hydrogen bonding consists of two intramolecular hydrogen bonds parallel to the glycosidic linkage for both chains, and two intralayer hydrogen bonds. The layers are packed hydrophobically. All hydroxymethyl group are positioned in the tg conformation. For the cellulose II form it was found that, in contrast to what seemed to emerge from the X-ray fibre diffraction data, both independent chains had the gt conformation. This idea already existed because of elastic moduli calculations and 13C-solid state NMR data. Recently, the structure of cellotetraose was determined. There appear to be a striking similarity between the structure obtained from the MD simulations and this cellotetraose structure in terms of packing of the two independent molecules, the hydrogen bonding network and the conformations of the hydroxymethyl group, which were also gt for both molecules. The structure forms a 3D hydrogen bonded network, and the contribution from electrostatics to the packing is more pronounced than in case of the Ibeta structure. In contrast to what is expected, in view of the irreversible transition of the cellulose I to II form, the energies of the Ibeta form is found to be lower than that of II by 1 kcal mol(-1) per cellobiose.

Conformations and internal mobility of a glycopeptide derived from bromelain using molecular dynamics simulations and NOESY analysis.

The conformation and internal flexibility of a glycopeptide Manalpha1-6 (Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAcbeta1-N(Asn-Glu-Ser-Ser), prepared from pineapple stem bromelain, have been analyzed using a combination of molecular dynamics (MD) simulations in water with NOESY 1H NMR spectroscopy. Theoretical NOESY cross-peak intensities were calculated by the CROSREL program on the basis of models, obtained from MD simulations, using a full relaxation matrix approach. Special attention was paid to the description of internal flexibility of the hexasaccharide moiety by the use of generalized order parameters, in combination with the application of an individual rotation correlation tme for each monosaccharide residue. The tetrapeptide moiety appeared to be very mobile during the MD simulations, which was confirmed by the absence of NOE cross peaks. For the oligosaccharide part a model was developed to estimate characteristic times for large reorientational motions around the glycosidic linkages, associated with conformational transitions. For the Manalpha1-6Man and the Fucalpha1-3GlcNAc linkages such a flexibility was found with a characteristic time of 2 ns. In contrast, the Xylbeta1-2Manbeta1-4GlcNAcbeta1-4GlcNAc part of the glycan appears to be relatively rigid.

Conformational analysis of the xylose-containing N-glycan of pineapple stem bromelain as part of the intact glycoprotein.

The conformational behavior of the N-glycan Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta of stem bromelain as part of the intact glycoprotein was investigated and compared with that of the same N-glycan as part of a bromelain-derived glycopeptide. Proton chemical shifts of the glycoprotein N-glycan were determined by 2D HOHAHA and 2D NOESY measurements, making use of the glycopeptide 1H NMR data. During each 2D NMR experiment about 4% of the glycoprotein denatured. Experimental data concerning interproton distances of the intact glycoprotein N-glycan were obtained by NOESY 1H NMR spectroscopy. Several theoretical models for the N-glycan, obtained by molecular dynamics simulations of the glycopeptide, were investigated. Comparison of experimental and theoretical NOESY cross peak intensities was performed with the program CROSREL. In comparison with the glycopeptide, the distribution of populations between two main conformations of the Fuc alpha 1-3GlcNAc linkage was altered. In addition, the omega = 60 degrees (gt) rotamer of the Man alpha 1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the omega = -60 degrees (gg) conformation exists exclusively. Except for the Xyl beta 1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fuc alpha 1-3GlcNAc and Man alpha 1-6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fuc alpha/Man alpha side of the N-glycan. A qualitative analysis of the NMR spectra showed a larger degree of mobility in the denatured glycoprotein N-glycan than in the intact glycoprotein.

Conformational analysis of methyl beta-cellobioside by ROESY NMR spectroscopy and MD simulations in combination with the CROSREL method.

Methyl beta-cellobioside has been studied extensively by molecular dynamics (MD) simulations in water and by ROESY NMR spectroscopy in order to establish its solution structure. The MD simulations were started with four significantly different minimal energy conformations. The MD trajectories were analysed with respect to interproton distances and mobility, in order to find models for application in the analysis of NMR data. The ROESY spectra were analysed by using the CROSREL method, which allows quantitative analysis of ROESY spectra through correction for the offset dependence and incorporation of HOHAHA transfer estimates. These results were compared with data obtained from an initial rate analysis of the ROESY data and with the MD data. It is concluded that methyl beta-cellobioside in aqueous solution is in the same extended conformation that is also found in the solid state.

A 1H-NMR and MD study of intramolecular hydrogen bonds in methyl beta-cellobioside.

The existence of an HO-3...O-5' intramolecular hydrogen bond in methyl beta-cellobioside in solution in Me2SO-d6 and H2O-CD3OD (4:1 w/w) was studied by 500-MHz 1H-NMR spectroscopy and MD simulations. Temperature coefficients for the chemical shift of the hydroxyl resonances in these solvents were determined and the rates of proton exchange in the latter solvent were obtained from NOE data. With H2O-CD3OD as the solvent, the HO-3...O-5' hydrogen bond was insignificant, but its presence in Me2SO-d6 was confirmed.

Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential.

The conformation is described of the sialyl alpha(2----3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----N)Asn and NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----N)Asn is presented and for each glycosidic linkage, except for the alpha(2----6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1----4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2----3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2----6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2----6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta structural element. This provides an explanation for the shift effects induced by alpha(2----6) attachment of NeuAc to the N-acetyllactosamine unit.