Tilt series

The unit cell and the Bravais lattice type for IM-5 were obtained from tilt series of SAED patterns such as that shown in Figure 2 (a = 14.3 A, b = 57.4 A, c - 20.1 A with... 

The scaling factor of 40 kHz was independently determined at — 60 °C from the powder spectrum of a random dispersion of a deuteriated PM sample59. This value was furthermore confirmed in the simulations of the 2H NMR tilt series for each of the three labeled methyl groups, according to the total spectral width and line-shape arguments discussed by Ulrich and Watts58. 

The analysis of a full tilt series of 2H NMR spectra not only allows the determination of the unique bond angle for a deuteriated methyl group, but also provides an internal check for the consistency of the spectral interpretation. In particular, simulations provide a means for the analysis of line-broadening effects, which arise from the sample mosaic spread as well as the intrinsic line width of the nuclear transition and instrumental factors. When line shapes are fitted to a full tilt series of spectra in a concerted manner and are also compared with the powder spectrum of an unoriented sample, the different contributions can be discerned. In that way an intrinsic line width of around 2 kHz is found for the spectra shown here, together with a mosaic spread between 8° and 10° for the three samples. 

From the good fit to the experimental spectra it is thus evident that one characteristic angle 9 describes the whole tilt series and that the underlying cone model for the bond vectors is consistent. The accuracy of the angles determined is estimated to be within... 

FIGURE 44. Tilt series of 2H NMR spectra from the deuteriomethyl groups Cl 8 (left column) and C20 (right column) of retinal in oriented purple membranes, at seven different sample inclination (a) in the spectrometer field. The line-shape simulations are superimposed over the experimental spectra in order to illustrate the good line fit obtained by the prediction method. Reprinted with permission from Reference 57. Copyright (1994) American Chemical Society... 

FIGURE 45. Orientation and conformation of retinal in bR, constructed from the individual methyl group orientations that have been determined by solid-state 2H NMR. The angles 6 of the C—CD3 bond vectors with respect to the membrane normal (N) were evaluated for Cis (37°), C49 (40°) and C20 (32°) from the zero-tilt spectra shown in Figure 44 and with the aid of line-shape simulation of the tilt series in Figure 42 and 43. Reprinted with permission from Reference 57. Copyright (1994) American Chemical Society... 

Figure 48 shows representative experimental 2H NMR spectra from the labeled retinal in bR in a dark-adapted PM sample. The line shape simulations that were generated in the data analysis are superimposed on the experimental spectra. The powder pattern [Figure 48(a)] serves as a general reference for the tilt series of spectra recorded at various sample inclinations [Figure 48(b)], because it defines the accessible frequency region over which the spectral intensity can occur. The oriented sample was measured at every 22.5° between 0° and 90°, of which three inclinations are represented in Figure 48(b) with a = 0°, 45° and 90°. 

FIGURE 48. Representative 2H NMR spectra (full lines) of dark-adapted bR (90 mg) containing deuteriated retinal, with line shape simulations (dashed lines) superimposed. Both the powder spectrum (a) from randomly oriented PM patches and the tilt series (b) over sample inclinations, a = 0, 45° and 90°, were recorded at — 60 °C (number of scans, 1.7 x 105, for a = 0°). Spectrum (c) was measured at 21 °C with a = 0° (number of scans, 3 x 105). Reprinted with permission from Reference 60. Copyright (1997) American Chemical Society... 

Three tilt series of ED patterns, tilted along the (h 0 0), (0 0 1) and (2h -h 0) axes, were collected on a JEOL 2000FX electron microscope (Fig. 1). Due to the limited specimen tilt angles of the microscope ( 45°), several crystals with different orientations are used for collecting a complete ED patterns. From these tilt series, 13 zone axes that contained strong and/or many reflections were chosen for 3D reconstruction [001], [010], [011], [012], [013], [014], [021], [023], [120], [121], [122], [241] and [5 18 0]. 

Firstly, the 3D indices of two reflections in the Fourier transform of each image are determined from the 3D reciprocal space reconstructed from three tilt series of electron diffraction (Fig. 1). Then all reflections in the Fourier transform of the image are converted from the indices of these two reflections into 3D indices in CRISP. The corresponding symmetries are imposed on amplitudes and phases. 

In the case of P Cu-phthalocyanine (space group P2i/c) only 8 different zones were found at 100 kV via SAED using a rotation-tilt holder (a=14.50 A, b= 4.70 A, c= 19.32 A, b=123°). With a rotation-double-tilt holder and in nano diffraction mode it was possible to detect 17 different zones at 300 kV (initial zone [001] and tilt about a and b ) from one crystal at room temperature (a=14.50A, b=4.84A, c=19.4lA, p=120.3°). In Fig. 5 the initial zone (middle) and two other zones obtained through different tilt series (a left hand side b right hand side) are shown exemplarily. 

Based on a Vainshtein plot, where the d values for the non tilt axis is plotted according to the tilt angle, the third axis and the one missing angle can be derived from each tilt series [10]. 

During the tilt about the main axes, 90° angles were preserved and the tilt series turned out to be symmetrical, which implies an orthorhombic system in each case. The third axes were calculated as 23.5 A and 25.3 A, respectively. These preliminary cell parameters have been refined according to the ratio of the axes in every measured zone leading to a higher cell parameter precision for both modifications (Mod I a=23.83 A, b=9.10 A, c=5.03 A Mod II a=10.22 A, b=25.43 A, c=4.I8 A). With the refined cell parameters it was possible to index the two-phase diffraction diagram. 

Table 1. Data for tilt series of aerinite Tilt about c ...

The three-dimensional electron tomographical construction of silica-supported metallocene catalysts using conventional TEM (Steinmetz et al 2000), and a novel method for the automated acquisition of tilt series for electron tomography of nanoparticles using STEM have been reported (Zeisse et al 2000). The HAADE-STEM is shown to be capable of determining the compositions of individual nanoparticle catalysts of a few atoms supported on porous substrates (Vaughan et al 1999). 

The protein structure is not known at high-resolution for any P-type pump, as crystals of a size and quality suitable for X-ray analysis have not yet been produced. However, the discovery that ordered two-dimensional semicrystalline arrays of ATPase molecules can be formed in situ in the membrane by incubation with tightly binding inhibitors such as vanadate (Skriver et al., 1981 Dux and Martonosi, 1983) has been of great help in obtaining a relatively detailed picture of the overall shape of the protein by electron microscopy. Fourier analysis of tilt series of electron micrographs permitted 3D-reconstructions of the Na+-K+- and Ca2+- ATPase protein structures to a resolution of 20-25 A (Figure 12a). Recently, this type of reconstruction analysis has been taken a step further to a resolution of about 14 A, and detailed structural features have become visible, even for the transmembrane domain (Toyoshima et al., 1993). The model of the Ca2+-ATPase peptide shown in... 

Tomographic tilt series and remote control of microscope 99-102... 

Record a single axis tilt series of particles embedded in negative stain, with a tilt range from -60" to +60". 

A recent three-dimensional reconstruction from a tilted series of negatively stained and of metal-shadowed preparations of the S-layer of Methanoplanus limicola[ Q6 has shown P6 symmetry, a lattice constant of 14.7 nm, and a thickness of 4.5 mn. Chemical analysis of these S-layers revealed an apparent molecular weight of the protein of 135 kDa, and the neutral sugar content of 240 mg/g polypeptide corroborates earlier suggestions... 

The three-dimensional reconstruction from a tilt series of a negatively stained cell envelope revealed a dome-shaped morphological imit with a wide opening toward the cytoplasmic membrane and a narrow opening to the exterior at the apex [125]. This design resembles that of Haloferax volcanii [121] and, to a lesser extent, that of Sulfolobus... 

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