Anaemia in juvenile chronic arthritis

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22121 or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 A and show that the asymmetric unit is the same in both crystal types.     

Two-dimensional crystallization of Ca-ATPase by detergent removal

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.     

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.     

Microstructure of stainless steel single-crystal electron beam welds

Previously developed techniques by the authors for the microstructural analysis of welds, that included the effects of both the growth crystallography and the weld pool shape, are applied to several cases involving the single-crystal electron beam welding of an Fe-15Ni-15Cr alloy. This evaluation of weld microstructures and associated dendritic growth patterns is based on a three-dimensional (3-D) geometrical analysis. The present study includes examination of the effects observed in overlapping, multipass autogenous welds and butt welds of two single crystals with different orientations, as well as effects due to variations in the welding speed. Weld pool shapes were found to change significantly with increasing welding speed—becoming narrower in cross section but more elongated in the welding direction. Additionally, all electron beam welds showed evidence of a plateau region in the center of the weld pool. The pool shapes, however, were found to be independent of the crystallographic orientation. Therefore, it is possible to extend the pool shape results to crystals welded in any orientation and even to polycrystals. The over-lapping multipass welds showed remarkable reproducibility from pass to pass and duplicated the structural patterns found in single-pass welds. The similarity in dendritic patterns within each pass indicated that the weld pool shapes were identical in all of the passes. The micro-structure of butt welds of two single crystals with different relative orientations showed a remarkable relationship to that associated with each individual crystallographic orientation, and the micro structure was, in effect, simply a composite of two single-pass microstructures. Additional microstructural details were also examined. The tendency toward branching of dendrites was associated with the transition from one dendrite growth orientation to another. It was also found that the nonpenetrating welds exhibited a small protrusion at the bottom of the weld. It is suggested that the modeling of weld pool shapes can be directly evaluated by comparing the predicted dendritic growth patterns based on the modeled shapes with the actual experimentally observed dendritic growth patterns. Formerly Visiting Scientist, Solid State Division, Oak Ridge National Laboratory.     

Distortion correction of tubular crystals: improvements in the acetylcholine receptor structure

Biological molecules often crystallize either as tubes, having helical symmetry, or as two-dimensional sheets. Both sorts of crystal are potentially suitable for structure determination to atomic resolution by electron crystallography, but their lattice distortions must first be corrected. We have developed a procedure for tubular crystals, based on independent alignment of very short segments against a reference structure, that allows accurate determination and correction of distortions in all three dimensions. Application of this procedure to images used previously to determine the 9 A structure of the acetylcholine receptor showed that about half of the signal loss caused by the distortions arises from effects correctable in the image plane (bending, changes in scale) and half from effects requiring out-of-plane correction (variations in tilt and in twist around the tube axis). By dividing the tubes into short segments (of lengths about equal to their diameter) it became possible to recover almost all of this loss without reducing appreciably the accuracy in the segmental alignments. The signal retention improved by only 10% at low resolution (20 A), but by progressively greater amounts at higher resolutions, up to approximately 40% at 9 A. As a result the finer structural details were more clearly resolved. With images of better electron-optical quality, much greater gains in signal retention should be obtained.     

New insights into the widmanstätten proeutectoid ferrite transformation: Integration of crystallographic and three-dimensional morphological observations

The crystallography and three-dimensional (3-D) morphology of Widmanstätten proeutectoid ferrite precipitates are examined in an Fe-0.12 wt pct C-3.28 wt pct Ni steel isothermally reacted at 650 °C, 600 °C, and 550 °C. This article integrates new orientation mapping (OM) results with the findings of a companion article to this one on the 3-D morphology of proeutectoid ferrit^[1] and an earlier transmission electron microscopy (TEM) study which is reanalyzed here in light of the new OM and 3-D results. All of these studies were performed for the same alloy and heat treatments. The 3-D morphologies and distributions of proeutectoid ferrite precipitates are now known to often be quite different from those deduced by conventional two-dimensional (2-D) microscopy techniques. The present crystallographic studies indicate that “primary” ferrite (nucleated directly on prior austenite grain boundaries) forms monolithic single crystals and can be approximated as elongated triangular pyramids. “Secondary” ferrite morphologies can be described as laths and plates branching into the austenite from a thick and/or broad allotriomorphic ferrite base. These secondary Widmanstätten branches are composed of many misoriented crystals with ferrite: ferrite boundaries between them and appear to approach a common orientation as they extend into the austenite grain. Implications of the current findings on existing growth and crystallography models are discussed, and a preliminary hypothesis or mechanism of ferrite formation has been proposed to account for the present observations.     

The formation mechanism(s), morphology,and crystallography of ferrite sideplates

The formation mechanism(s), morphology, and crystallography of secondary ferrite sideplates were investigated with transmission electron microscopy (TEM), scanning electron microscopy (SEM), electron backscatter pattern (EBSP) analysis, and optical microscopy in a high-purity Fe—0.12 wt pct C—3.3 wt pct Ni alloy isothermally transformed at temperatures of 550 °C, 600 °C, 650 °C, and 675 °C. The results indicate that two different mechanisms contribute to the formation of these sideplates at austenite grain boundaries. On the first mechanism, primary sideplates form initially, followed by rapid lateral impingement along their bases, resulting in a region along the grain boundary which very early in the growth process resembles an allotriomorphic film. On the second mechanism, sympathetic nucleation of ferrite sideplates occurs atop pre-existing ferrite allotriomorphs, resulting in ferrite:ferrite grain boundaries and significant crystallographic misorientations between the sideplates and the allotriomorphs with which they are associated. These results indicate that “secondary sideplates” and the allotriomorphs from which they evolve arenot composed of monolithic single crystals formed by a morphological instability mechanism but are instead composed of multiple crystals formed by individual nucleation events. Previous investigations in Ti-Cr alloys and a high chromium stainless steel suggest that the findings presented here may be applicable to a number of other alloy systems as well. This article is based on a presentation made during TMS/ASM Materials Week in the symposium entitled “Atomistic Mechanisms of Nucleation and Growth in Solids,” organized in honor of H.I. Aaronson’s 70th Anniversary and given October 3–5, 1994, in Rosemont, Illinois.     

Measurement of Particle Movement in Granular Soils Using Image Analysis

The flowing behavior of granular soils in the form of localized deformations and shear bands is a difficult phenomenon to define explicitly in the modeling of soil-structure interface problems. However, for development of accurate numerical solutions for these problems, an estimation of particle movements is important in determining the nature of displacement fields within the granular media. Video images from direct shear tests were used to capture the movement of individual grains relative to the movement of a ribbed structural surface. Two different algorithms have been developed to determine particle displacements in an image sequence of the interface during a direct shear test. BMAD employs a block-matching algorithm using unit patterns of images to determine two-dimensional particle displacement vectors. MATCH, on the other hand, computes displacements based on centroid locations of segmented particles that are matched using a four-level filter algorithm in successive image frames. Measured hardware dependent noise was reduced during image acquisition by means of a frame averaging technique implemented in BMAD. BMAD was successfully employed to determine particle displacements in Ottawa sand images obtained during shearing on a ribbed surface. The results were verified with MATCH using the same image frames.     

Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A

Three photosynthetic complexes, light-harvesting complex 2 (LH2), light-harvesting complex 1 (LH1), and the reaction centre-light-harvesting complex 1 photounit (RC-LH1), were purified from a single species of a purple bacterium, Rhodobacter sphaeroides, and reconstituted into two-dimensional (2-D) crystals. Vesicular 2-D crystals of LH1 and RC-LH1 were imaged in negative stain and projection maps at 25 A resolution were produced. The rings formed by LH1 have approximately the same mean diameter as the LH1 rings from Rhodospirillum rubrum ( approximately 90 A) and therefore are likely to be composed of 15 to 17 alphabeta subunits. In the projection map calculated from the RC-LH1 2-D crystals, the reaction centre is represented by an additional density in the centre of the ring formed by the LH1 subunits. The marked improvement of shape and fine structure after a rotational pre-alignment of the RC-LH1 unit cells before averaging strongly suggests that the RC is not in a unique orientation within the LH1 rings. Tubular crystals of LH2 showed a high degree of order and allowed calculation of a projection map at 6 A resolution from glucose-embedded specimens. The projection structure shows a ring of nine alphabeta subunits. Variation of the alpha-helical projection densities suggests that the 9-fold symmetry axis is tilted with respect to the membrane normal.     

Correspondence of left ventricular ejection fraction determinations from two-dimensional echocardiography, radionuclide angiography and contrast cineangiography

OBJECTIVES: This study assessed the agreement of left ventricular ejection fraction determinations from two-dimensional echocardiography, radionuclide angiography and contrast cineangiography. BACKGROUND: Previously published reports suggest that two-dimensional echocardiography, radionuclide angiography and contrast cineangiography are equally acceptable methods of assessing left ventricular ejection fraction on the basis of high coefficients of correlation. However, correlation of methods does not necessarily imply agreement. METHODS: In a prospective analysis, 25 consecutive subjects all had two-dimensional echocardiography and radionuclide angiography performed within 10 days of each other in the cardiology department of metropolitan community hospital. A retrospective computer search (Medline) revealed seven studies, using the coefficient of correlation (r), comparing two-dimensional echocardiographic left ventricular ejection fraction (n = 268) with radionuclide angiographic (n = 174) or contrast cineangiographic (n = 119) left ventricular ejection fractions. RESULTS: The eight individual studies (n = 293) comparing two-dimensional echocardiography with either radionuclide angiography or contrast cineangiography exhibited coefficients of correlation ranging from 0.78 to 0.93. Agreement analysis using the method of Bland and Altman was performed by averaging the results obtained from the two techniques and determining how disparate any single ejection fraction was (with 95% confidence limits) from the mean value. Agreement ranged from 23% to 42% around the mean ejection fraction. The average lack of agreement between the two methods for all studies involved was 17%, with an average r value of 0.86. CONCLUSIONS: Left ventricular ejection fraction determinations by means of two-dimensional echocardiography, radionuclide angiography and contrast cineangiography exhibit high correlation and only moderate agreement. High correlation does not always imply high agreement. These results suggest that, when validated by agreement analysis, multiple studies may not be necessary in appropriate clinical situations, potentially reducing costs.     

Lupus erythematosus profundus associated with neonatal lupus erythematosus

Overproduction and crystallization of Bacillus stearothermophilus ribosomal protein S7 (BstS7), a primary 16S rRNA binding protein and also a translational repressor protein, have been performed to analyze its three-dimensional structure by X-ray crystallography. Ribosomal protein BstS7 was expressed in the cytoplasmic fraction of the E. coli cells and purified to homogeneity. This recombinant BstS7 was used to produce crystals with P2(1) symmetry that diffracted to 2.5 A resolution which are suitable for high-resolution X-ray crystallographic analysis.     

A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli

We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.     

Purification and crystallization of benzoylformate decarboxylase

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.     

The martensitic phases and their stability in CuZn and CuZnAl alloys—III. The transformation between the high temperature phase and the 2H martensite

The stability between the high temperature β phase and the 2H martensite is analyzed in CuZnAl single crystals of a high electron concentration around 1.52. The results are compared with those deduced from previous reports, and the equilibrium temperatures and entropy differences are determined. A phenomenological model for the stress induced transformation explains well the observed crystallography.     

Use of misorientation values to further understand deformation in rail steels

Abstract

The high contact stresses between a railway wheel and rail result in local plastic deformation in both the rail and wheel steel. Optical metallography and hardness testing have traditionally been used to quantify the extent of deformation present; however, these methods only give limited information about the deformation mechanism and the role of microstructure. In this study, electron backscattered diffraction has been used to assess the depth and degree of deformation using the kernel average misorientation function; where the crystallographic misorientation between and within pearlite colonies has been quantified using a local average misorientation function. This technique gives invaluable crystallographic information about the deformed microstructure to aid understanding of the deformation mechanism. The application of the kernel average misorientation function has been modelled for idealised rail microstructures, including after the simulation of deformation via shear, in order to understand how the average kernel average misorientation values are developed under the high levels of deformation seen in rail steels.     

Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (Aad-Cys-Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging-drop vapour-diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X-rays to at least 0.11-nm resolution at the European Synchrotron Radiation Facility and belong to the space group P2(1)2(1)2(1), with unit-cell dimensions a = 4.68, b = 7.15, c = 10.10 nm. Their asymmetric unit contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X-rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3(1)21, with unit-cell dimensions a = 10.10, b = 10.10, c = 11.567 nm. Their asymmetric unit also contains a single IPNS.Fe(II).Aad-Cys-Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well-ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X-ray-absorption fine-structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe-S(Aad-Cys-Val) bond length of 0.234 +/- 0.003 nm.     

Simplified Plane-Strain Modeling of Stone-Column Reinforced Ground

The acceleration of consolidation rate by stone columns was mostly analyzed within the framework of a basic unit cell (i.e., a cylindrical soil body around a column). A method of converting the axisymmetric unit cell into the equivalent plane-strain model would be required for two-dimensional numerical modeling of multicolumn field applications. This paper proposes two simplified conversion methods to obtain the equivalent plane-strain model of the unit cell, and investigates their applicability to multicolumn reinforced ground. In the first conversion method, the soil permeability is matched according to an analytical equation, whereas in the second method, the column width is matched based on the equivalence of column area. The validity of these methods is tested by comparison with the numerical results of unit-cell simulations and with the field data from an embankment case history. The results show that for the case of linear-elastic material modeling, both methods produce reasonably accurate long-term consolidation settlements, whereas for the case of elastoplastic material modeling, the second method is preferable as the first one gives erroneously lower long-term settlements.     

Three-dimensional map of the plasma membrane H+-ATPase in the open conformation

The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cell's plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200mV. Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 A, this map reveals ten membrane-spanning alpha-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands.     

Scanning electron microscopy and microcrystals in articular diseases

The value of scanning electron microscopy (SEM) in the study of crystals in articular diseases is underlined in several cases of examination of joint fluid, or crystal deposits in articular or periarticular tissues obtained by percutaneous or surgical treatment with chemical and crystallographic correlations. Apatite crystals. Two deposits of hydroxyapatite of the rotator cuff were studied by SEM, crystallographic techniques and chemical analysis. SEM study showed spherical aggregates of various size. Urate crystals. Three tophi were observed by SEM, with crystallographic techniques and chemical analysis. Their needle-shape and their great size (20 m) were characteristic. Calcium pyrophosphate crystals. In a case of typical clinical and radiological features, examination of joint fluid, with chemical correlation showed shorter and thicker crystals than those or urate. The precise identification of crystals is based on sophisticated crystallographic techniques such as X-ray diffraction, although SEM allows an accurate and quite simple morphologic study, most often sufficient.