Structure determination of tubular crystals of membrane proteins. II. Averaging of tubular crystals of different helical classes

A set of programs has been developed for averaging the data from tubular crystals belonging to different helical classes. This was done either by (i) cutting out molecules constituting a unit cell from density maps, and aligning and averaging them in real space; (ii) transforming the densities in a unit cell to layer-line data according to a (possibly artificial) helical symmetry, aligning and averaging them in reciprocal space. These methods were applied to tubular crystals of Ca2+-ATPase. Either method worked well and substantially improved the data quality. Transforming the reconstructed images to the layer-line data has many advantages and is essential for fully exploiting the power of averaging.     

Structure determination of tubular crystals of membrane proteins. IV. Distortion correction and its combined application with real-space averaging and solvent flattening

A method for correction of three-dimensional distortions has been developed for helical assemblies and applied to tubular crystals of Ca2+-ATPase. This method approximates distorted helical particles with short straight segments of different orientation parameters, which are determined by fitting them to the reference data in reciprocal space. Thus, the method follows Beroukhim and Unwin [Ultramicroscopy 70 (1997) 57], but is more extended to achieve better alignment and to cope with images of poor S/N ratio. Substantial improvements were achieved by dividing the reference image into the segments of optimal lengths in exactly the same way as the test, and treating the distortions in the near and far sides of a helical particle separately. The improvement was further enhanced when combined with real-space averaging [Yonekura, Toyoshima, Ultramicroscopy 84 (2000) 15] and solvent flattening [Yonekura, Toyoshima, Ultramicroscopy 84 (2000) 29], and most pronounced when all these three were applied iteratively.     

Structure determination of tubular crystals of membrane proteins. I. Indexing of diffraction patterns

Indexing of diffraction patterns is the starting point of every crystallographic analysis. The diffraction patterns from helical particles consist of a series of layer-planes, the indexing of which involves the assignment of the start numbers of the helices that contribute to the layer-planes. The indexing could be challenging if the diameter of the helical particle is large and if the conventional indexing methods based on selection rule is used. Presented here is a tutorial on how to index them using (h, k; n) indexing method, which is particularly useful for tubular crystals of membrane proteins.     

Electron-irradiation-induced flattening of negatively stained 2D protein crystals

The thickness of negatively stained 2D crystalline arrays of the bladder membrane does not vary significantly during air drying and exposure to high vacuum. High-dose electron irradiation reduces the thickness to about 60% of the native value. These results, together with the fact that the same behaviour has been observed on another 2D system (gap junctions), indicate that the flattening induced by an electron beam on 2D crystals may be general. The implications for 3D reconstruction of negatively stained objects are discussed.     

Capacitance determination of the area thermal expansion of dielectric crystals

Both the area thermal expansion and the dielectric constant of a solid insulator are determined by a capacitance technique in which the change in area of a capacitor is first caused by the variation with temperature of the dimensions of the dielectric, and then by the variation over the same temperature range of the dimensions of copper electrodes, the same sample being used in both cases. This method is particularly convenient for small, flat crystals under hydrostatic pressure and for anisotropic crystals. It is believed that a similar method could be used to measure the compressibility. The apparatus and calculations are described, and the results of a test run on the thermal expansion of NaCl are compared with previous data. Between 77 and 250 K, the measured linear thermal expansion L (T)/L (300) of NaCl agrees with the results of a Fabry-Perot etalon experiment to within 0.03%, giving differences in the coefficient of linear expansion of 10% in the worst case.     

The thickness determination of organic crystals under low dose conditions using electron energy loss spectroscopy.

In the 3-dimensional (3-D) reconstruction of protein crystals with variable thicknesses the electron images and diffraction patterns can only be merged if the crystal thickness is known. Measurement of the thickness using the ratio of the number of inelastically scattered electrons to the number of electrons in the zero loss peak can be accomplished with parallel electron energy loss spectrometry (PEELS). A theoretical analysis of the accuracy of the technique on paraffin crystals of different thicknesses is presented. Our experimental studies with paraffin crystals show the feasibility of measuring a single layer of 47A with good accuracy under low dose and low temperature conditions. A simple experimental apparatus is proposed to obtain thicknesses from small regions of unstained protein crystals prior to collecting the 3-D data sets from the unexposed area of the same crystal.     

Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona.

Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.     

Electron microscopy of tRNA crystals. I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNAphe. The thickness of the thinnest was estimated at 130 A corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

Automatic size determination of membrane vesicles by freeze-fracture electron microscopy

In biological processes involving membrane vesicles, the vesicle sizes and the size distribution of the vesicle population are usually important process parameters. A previous method for determining these parameters by freeze-fracture electron microscopy exclusively makes use of concave profiles with half-cast shadow, the so-called equatorial profiles. This method suffers from two disadvantages: (i) the measurement is done manually and is therefore subjective, and (ii) only a small subpopulation of the profiles, those showing half-cast shadow, can be used. We describe an automatic, computerized method with which the vesicle size can be calculated from any concave profile with cast shadow. This method is based on the relationship between the angle of shadowing and the surface areas of the profile and the profile's uncoated region. Since the vesicles are processed automatically, the procedure is reproducible and not biased by subjective measurements. The method was tested both on synthetic, computer-generated profiles and on large unilamellar vesicles. It allows for the use of about four times as many profiles as the equator method in determining the vesicle size distribution. This means that the distribution is more reliable thanks to the larger population used for its determination. Furthermore, the reliability of our method is exemplified by its stability concerning errors in its parameters.     

Electron microscopy of tRNA crystals: I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNA^phe. The thickness of the thinnest was estimated at 130 Å corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

管状电热膜元件工业化热喷镀过程中传输方案的反求工程设计

本文应用功能反求分析原理对管状电热膜元件在工业化热喷镀过程中的传输方式进行了反求工程设计。     

Structure determination at the atomic level from dynamical electron diffraction data under systematic row conditions.

We discuss a method to obtain structural information on crystals at the atomic level in high-resolution transmission electron microscopy from dynamical diffraction data under systematic row conditions. Working at a fixed incident energy and within an N-beam approximation, data is required at a well defined set of N incident beam orientations to determine the scattering matrix, one orientation for each column in the matrix. At each orientation the corresponding column of the scattering-matrix is obtained by Fourier transformation of the exit surface wave function. Thus, in addition to each exit surface image, we must recover the phase of the wave function for that orientation in the image plane. We show that retrieval of the phase using algorithms based on conservation of flux, which assume continuity of the phase, can yield incorrect solutions for the phase. This is because singularities can occur in the phase of the wave field at points where the intensity is zero, which can lead to edge dislocations in the phase. We demonstrate, using a model example, how these edge dislocations arise. We will show that phase retrieval from a through focal series of measurements or using the Gerchberg-Saxton algorithm (starting from measurements of an image and the corresponding diffraction pattern), correctly retrieves the phase and hence the exit surface wave function for all the orientations required to obtain the scattering-matrix. The dynamical (multiple) scattering can then be inverted to uniquely obtain the projected potential.     

采用裂解气相色谱/质谱法研究双酚A环氧树脂结构

采用裂解气相色谱/质谱联用方法对不同品种和牌号的双酚A环氧树脂进行分析研究。选择550℃裂解,其裂解产物通过SE～54高效毛细管柱分离和质谱鉴定,提供了有关环氧树脂的特征结构信息。通过对总离子流图中裂解碎片峰峰面积比值的计算,可初步鉴定7种不同牌号的环氧树脂。此方法,具有样品无需前处理,作量少,对应性强,灵敏度高等特点。     

Influence of calcium on direct incorporation of membrane proteins into in-plane lipid bilayer

Reconstitution of transmembrane proteins by direct incorporation into supported lipid bilayers (SLBs) is a new method to provide suitable samples for high-resolution atomic force microscopy (AFM) analysis of membrane proteins. First experiments have reported successful incorporation of proteins into detergent-destabilized SLBs. Here, we analyzed by AFM the incorporation of membrane proteins in the presence of calcium, a divalent cation functionally important for several membrane proteins. Using lipid-phase-separated membranes, we first show that calcium strongly stabilizes the SLBs decreasing the insertion of low cmc detergents, dodecyl-beta-maltoside, dodecyl-beta-thiomaltoside, and N-hexadecylphosphocholine (Fos-Choline-16) and further insertion of proteins. However, high yield of protein insertion is recovered in the presence of calcium by increasing the detergent concentration in the solution. These data revealed the importance of the calcium in the structure of SLBs and provided new insights into the mechanism of protein insertion into these model membranes.     

Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX

We present a method for immunolabeling of multiple species of membrane proteins with high spatial resolution. It allows differentiation of equally sized very small markers with different chemical compositions, which leads to high labeling efficiency and reduces steric hindrance of closely spaced immunolabeled biomolecules. Markers such as CdSe/ZnS semiconductor quantum dots and colloidal gold particles are distinguished by differential contrast in high-angle annular detector dark-field STEM mode or by EDX microanalysis of their elemental contents. This method was tested by observation of labeled AMPA- and NMDA-type glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. To improve particle visibility and detectability, the replica films were made exclusively with carbon to avoid the high background of conventional platinum/carbon replica. Extension of the method is suggested by detection of 1.4 nm nanogold particles and its potential application in the biological imaging research.     

Dual colorimetric and fluorescent determination of iron (III) using a novel squaraine dye

A novel squaraine dye, 6-carboxy-2-[[3-[1,3-dihydro-3,3-dimethy-1-ethyl-2H-indol-2-ylidene)methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-ylidene]methyl]-3,3-trimethy-3H-indolium, has been synthesized. The squaraine dye was found to be a dual colorimetric and fluorescent probe for the determination of iron (III) in CH₃CH₂OH/H₂O (4:1, v/v) exhibiting high selectivity and sensitivity. The binding of squaraine dye +Fe³⁺ was studied by a Job’s plot and Fourier transform infrared and ¹H nuclear magnetic resonance spectroscopies. The result indicates that the squaraine dye may be utilized as a naked-eye and real-time probe for the efficient determination of Fe^3+.     

Structure analysis of small proteins by electron microscopy: valinomycin, bacitracin and low molecular weight cell growth stimulators.

Dark field electron microscopy was combined with optical filtering to study at high resolution the structure of the cyclopeptide antibiotics, bacitracin and valinomycin, and two proteins of unknown structure, LMW-CSA N and B, low molecular weight granulocyte colony stimulating activity isolated from medium conditioned with normal or leukemic leukocytes. For bacitracin and valinomycin the images faithfully represented the known structural features at a resolution of 0.5 nm or better, depicting a two-ring structure for bacitracin, as well as the position of the potassium ion in valinomycin. Both proteins of unknown structrue had at least one cyclic peptide portion. LMW-CSA N had a size of 2.0 nm, LMW-CSA B of 2.4 nm. A potential site of the calcium ionophoric activity in the latter protein was found to be in the larger of the two ring portions constituting the molecule.     

Aspects of scanning microdensitometry. III. The monochromator system.

Microdensitometric errors can result from various factors associated with the monochromator system, including imperfect monochromaticity of the light, incorrect setting of the wavelength, and non-uniform illumination of either the microscopic field or the objective aperture. Certain types of potential error are characteristic of particular instruments. Thus in the Vickers M85 microdensitometer, where the flying spot is the reduced image of a hole situated at the monochromator exit aperture, the interpretation of results obtained with different spot sizes is complicated by the fact that the hole size affects both the spatial resolution and the spectral bandwidth of the system. Similarly, in instruments in which the monochromator exit slit lies in an aperture plane the numerical aperture of the whole system may be affected by the spectral bandwidth and vice versa. Overall instrumental sensitivity is mainly limited at the blue and red ends of the spectrum respectively by the lamp output and the photomultiplier tube sensitivity. Quartz-iodine lamps are slightly brighter than conventional tungsten sources, especially at short wavelengths, but tend to be less stable photometrically and are more expensive. Simple refracting monochromators and graded-spectrum interference filters in general pass more light, in the visible spectrum, than do grating monochromators of similar bandwidth. Most errors of wavelength setting can be avoided by routinely measuring at that wavelength, lambda(max), found empirically to give the maximum absorbance or integrated absorbance. Off-peak wavelengths can be set reproducibly with the aid of an eyepiece spectroscope, or by adjusting the wavelength so that the absorbance of a given specimen is some precise fraction of that at lambda(max).     

Impact wear of MgO single crystals I. Wear characteristics

The impact wear characteristics of MgO single crystals have been investigated using an impact testing machine provided with a sintered alumina hammer having a tip radius of 2 mm. There is a fixed zero wear duration time for each selected impact load. Plastic deformation caused by slip of the {110} planes and cracking by impact occur during zero wear, but wear fragment are not produced. The zero wear period corresponds to a process of impact fatigue. Measurable wear, which is obtained by repeated impact over the zero wear limit, depends on the impact load and the action of wear fragments. When the test is performed in water wear is accelerated by abrasive erosion caused by impulsive flow of water containing wear fragments between the hammer and the scar wall.