A rhombohedral phase of lipid containing a membrane fusion intermediate structure

We constructed the electron density distribution from the x-ray diffraction of a phase of phospholipid that exhibited rhombohedral symmetry. To determine the phases of the diffraction amplitudes, we first extended the well-known one-dimensional swelling method for planar bilayers to a three-dimensional method applicable to a layered system containing in-plane structures, such as rhombohedral structures. The complete phase determination was accomplished by a combination of the swelling method and Luzzati's pattern recognition method. The constructed electron density distribution showed that in each unit cell, two apposed monolayers merged across the water layer and developed into an hourglass structure consistent with a postulated membrane fusion intermediate state called a stalk. The observation of the stalk structure lends a strong support to the stalk hypothesis for membrane fusion and opens a way to measure the structural parameters in the fusion pathway.     

In Situ D-periodic Molecular Structure of Type II Collagen

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Structural states of myelin observed by x-ray diffraction and freeze- fracture electron microscopy

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.     

Vitrosin: a member of the collagen class

Vitrosin, a fibrous protein obtained from the vitreous humor of the eye in the form of an indefinitely long fibril about 100 to 150 A in diameter, has been identified as a member of the collagen class of proteins. It is characterized by the collagen wide-angle x-ray diffraction pattern, and axial periodicity of about 640 A determined by electron microscopy and small-angle x-ray diffraction, an amino acid pattern characteristic of collagen as determined by paper chromatography, and a hydroxyproline and glycine content also typical of collagen. The glycine-hydroxyproline ratio is somewhat lower than that for most vertebrate collagens.     

Crystals of crotoxin suitable for high resolution x-ray diffraction analysis

The phospholipasic presynaptic neurotoxin, crotoxin, has been crystallized in a morphology suitable for single crystal x-ray diffraction analysis. The conditions for growth and the unit cell parameters (P4(1)22 or P4(3)22, a = b = 38.5 A, c = 256.9 A, 1 molecule/asymmetric unit) are similar to the very thin plate-like crystals which have been studied with electron diffraction and electron microscopy by Chiu and his colleagues (Jeng, T.-W., Chiu, W., Zemlin, F., and Zeitler, E. (1984) J. Mol. Biol. 175, 93 - 97). These two macroscopic crystal morphologies of what is likely to be a very similar, if not identical, lattice structure will permit the complementary application of electron diffraction/microscopy and x-ray diffraction to understanding the structural basis of the interactions between a phospholipasic neurotoxin and its membrane target.     

Crystalline structure of poly(hexamethylene adipate). Study on the morphology and the enzymatic degradation of single crystals

The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture.     

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.     

Single crystals of V-amylose complexed with alpha-naphthol

Lamellar square single crystals of V-amylose were obtained by adding alpha-naphthol to metastable dilute aqueous solutions of synthetic amylose chains with an average degree of polymerization of 100. The morphology and structure of the crystals were studied using low-dose transmission electron microscopy including high-resolution imaging, as well as electron and X-ray diffraction. The crystals are crystallized in a tetragonal P4(1)2(1)2 or P4(3)2(1)2 space group with unit cell parameters, calculated from X-ray diffraction data, a = b = 2.2844 nm (+/-0.0005) and c = 0.7806 nm (+/-0.001), implying the presence of two amylose chains per unit cell. High-resolution lattice images of the crystals confirmed that the amylose chains were crystallized as 8-fold helices corresponding to the repeat of four maltosyl units.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES ON A HOMOLOGOUS SERIES OF SATURATED PHOSPHATIDYLCHOLINES

Three homologous saturated phosphatidylcholines were studied by electron microscopy after tricomplex fixation. The results are compared with those obtained by x-ray diffraction analysis of the same and some other homologous compounds, in the dry crystalline state and after tricomplex fixation. By electron microscopy alternating dark and light bands are observed which are likely to correspond to phosphatide double layers. X-Ray diffraction reveals the presence of lamellar structures of regular spacing. The layer spacings obtained by both methods are in good agreement. From the electron micrographs the width of the polar parts of the double layers can be derived directly. The width of the carboxylglycerylphosphorylcholine moiety of the layers is found by extrapolating the x-ray diffraction data to zero chain length of the fatty acids. When from this width the contribution of the carboxylglyceryl part of the molecules is subtracted, again we find good agreement with the electron microscope measurements. An attempt has been made to account for the different layer spacings measured in terms of orientation of the molecules within the double layers.     

Direct determination of crystallographic phases for diffraction data from phospholipid multilamellar arrays.

Direct determination of crystallographic phases based on probabilistic of sigma 1 and sigma 2 "triplet" structure invariants has been found to be an effective technique for structure analysis with lamellar x-ray or electron diffraction intensity data from phospholipids. In many cases, nearly all phase values are determined, permitting a structure density (electron density for x-ray diffraction; electrostatic potential for electron diffraction) map to be calculated, which is directly interpretable in terms of known bilayer lipid structure. The major source of error is found to be due to the distortion of observed electron diffraction intensity data by incoherent multiple scattering, which can significantly affect the appearance of the electrostatic potential map, but not the success of the phase determination, as long as the observed Patterson function can be interpreted.     

The molecular structure of the high potential iron-sulfur protein isolated from Ectothiorhodospira halophila determined at 2.5-A resolution

The molecular structure of a high potential iron-sulfur protein (HiPIP) isolated from the purple photosynthetic bacterium, Ectothiorhodospira halophila strain BN9626, has been solved by x-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R value of 18.4% including all measured x-ray data from 30.0- to 2.5-A resolution. Crystals used in the investigation contained two molecules/asymmetric unit and belonged to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. An interpretable electron density map, obtained by combining x-ray data from one isomorphous heavy atom derivative with non-crystallographic symmetry averaging and solvent flattening, clearly showed that this high potential iron-sulfur protein contains 71 amino acid residues, rather than 70 as originally reported. As in other bacterial ferredoxins, the [4Fe-4S] cluster adopts a cubane-like conformation and is ligated to the protein via four cysteinyl sulfur ligands. The overall secondary structure of the E. halophila HiPIP is characterized by a series of Type I and Type II turns allowing the polypeptide chain to wrap around the [4Fe-4S] prosthetic group. The hydrogen bonding pattern around the cluster is nearly identical to that originally observed in the 85-amino acid residue Chromatium vinosum HiPIP and consequently, the 240 mV difference in redox potentials between these two proteins cannot be simply attributed to hydrogen bonding patterns alone.     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

Axial electron density of human scleral collagen. Location of proteoglycans by x-ray diffraction.

The low angle meridional x-ray diffraction pattern from fresh human sclera was analyzed to ascertain if collagen-bound proteoglycans affect the axially-projected electron density distribution to the same extent as appears to occur in the cornea. The results showed that, unlike cornea, the electron density of the sclera is similar to that seen in rat tail tendon collagen. The proteoglycans were specifically stained using either Cuprolinic blue or Cupromeronic blue, both under critical electrolyte conditions. The tissue was then examined by electron microscopy and by low angle x-ray diffraction. The electron-optical observations suggested that proteoglycans associate with collagen near the d/e staining bands in the gap zone. A difference Fourier analysis from the x-ray results confirmed that these observations were not e.m. preparative artefacts and allowed a quantitative estimate to be made of the axial extent of the proteglycans in the wet tissue.     

ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.     

Radial packing, order, and disorder in collagen fibrils.

Collagen fibrils resemble smectic, liquid crystals in being highly ordered axially but relatively disordered laterally. In some connective tissues, x-ray diffraction reveals three-dimensional crystallinity in the molecular packing within fibrils, although the continued presence of diffuse scatter indicates significant underlying disorder. In addition, several observations from electron microscopy suggest that the molecular packing is organized concentrically about the fibril core. In the present work, theoretical equatorial x-ray diffraction patterns for a number of models for collagen molecular packing are calculated and compared with the experimental data from tendon fibrils. None of the models suggested previously can account for both the crystalline Bragg peaks and the underlying diffuse scatter. In addition, models in which any of the nearest-neighbor, intermolecular vectors are perpendicular to the radial direction are inconsistent with the observed radial orientation of the principal approximately 4 nm Bragg spacing. Both multiple-start spiral and concentric ring models are devised in which one of the nearest-neighbor vectors is along the radial direction. These models are consistent with the radial orientation of the approximately 4 nm spacing, and energy minimization results in radially oriented crystalline domains separated by disordered grain boundaries. Theoretical x-ray diffraction patterns show a combination of sharp Bragg peaks and underlying diffuse scatter. Close agreement with the observed equatorial diffraction pattern is obtained. The concentric ring model is consistent with the observation that the diameters of collagen fibrils are restricted to discrete values.     

Crystallization of rat intestinal fatty acid binding protein. Preliminary X-ray data obtained from protein expressed in Escherichia coli

Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000. The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees. Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide. The crystals are remarkably resistant to x-ray damage. X-ray diffraction data have been observed to 2.0 A resolution. Platinum chloride was used to generate a potential isomorphous heavy atom derivative.     

Scanning X-Ray Nanodiffraction on Dictyostelium discoideum

We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions.     

New Insights into the Stratum Corneum Lipid Organization by X-Ray Diffraction Analysis

The characteristic 13-nm lamellar phase that is formed by lipids in the outermost layer of the skin, the stratum corneum (SC), is very important for the barrier function of the skin. To gain more insight into the molecular organization of this lamellar phase, we performed small-angle x-ray diffraction (SAXD) using various lipid mixtures mimicking the lipid composition in SC. In the SAXD pattern of each mixture, at least seven diffraction orders were observed, attributed to the lamellar phase with a repeat distance ranging from 12.1 to 13.8 nm. Using the sampling method based on the variation in repeat distance, we selected phase angles for the first six diffraction orders. Using these phase angles for the lamellar phase, a high-resolution electron density distribution could be calculated. Subsequently, from SAXD patterns of isolated SC, the electron density distribution of the lamellar phase was also calculated and appeared to be very similar to that in the lipid mixtures. This demonstrates that the lipid mixtures serve as an excellent model for the lipid organization in SC, not only with respect to the repeat distance, but also in terms of the electron density distribution within the unit cell.     

Characterization of a quasicrystalline phase in codispersions of phosphatidylethanolamine and glucocerebroside

Synchrotron x-ray diffraction, differential scanning calorimetry, and electron spin resonance spectroscopy have been employed to characterize a quasicrystalline phase formed in aqueous dispersions of binary mixtures of glucocerebroside and palmitoyloleoylphosphatidylethanolamine. Small- and wide-angle x-ray scattering intensity patterns were recorded during temperature scans between 20 degrees and 90 degrees C from mixtures of composition 2, 5, 10, 20, 30, and 40 mol glucocerebroside per 100 mol phospholipid. The quasicrystalline phase was characterized by a broad lamellar d-spacing of 6.06 nm at 40 degrees C and a broad wide-angle x-ray scattering band centered at approximately 0.438 nm, close to the gel phase centered at approximately 0.425 nm and distinct from a broad peak centered at 0.457 nm observed for a liquid-crystal phase at 80 degrees C. The quasicrystalline phase coexisted with gel and fluid phase of the pure phospholipid. An analysis of the small-angle x-ray scattering intensity profiles indicated a stoichiometry of one glucosphingolipid per two phospholipid molecules in the complex. Structural transitions monitored in cooling scans by synchrotron x-ray diffraction indicated that a cubic phase transforms initially into a lamellar gel. Thermal studies showed that the gel phase subsequently relaxes into the quasicrystalline phase in an exothermic transition. Electron spin resonance spectroscopy using spin labels located at positions 7, 12, and 16 carbons of phospholipid hydrocarbon chains indicated that order and motional constraints at the 7 and 12 positions were indistinguishable between gel and quasicrystalline phases but there was a significant decrease in order and increase in rate of motion at the 16 position on transformation to the quasicrystalline phase. The results are interpreted as an arrangement of polar groups of the complex in a crystalline array and a quasicrystalline packing of the hydrocarbon chains predicated by packing problems in the bilayer core requiring disordering of the highly asymmetric chains. The possible involvement of quasicrystalline phases in formation of membrane rafts is considered.