The dynamics of the layer-displacement fluctuations in smectic membranes have been studied by x-ray photon correlation spectroscopy (XPCS). We report transitions from an oscillatory damping regime to simple exponential decay of the fluctuations, both as a function of membrane thickness and upon changing from specular to off-specular scattering. This behavior is in agreement with recent theories. Employing avalanche photodiode detectors and the uniform filling mode of the synchrotron storage ring, the fast limits of XPCS have been explored down to 50 ns.
Negative staining can provide detailed, two-dimensional images of biological structures, but combining tomography with negative staining can provide three-dimensional images. Basic requirements for a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain is not degraded with the intensive electron dose needed to collect a full set of tomographic images. A commercially available, tungsten-based stain, methylamine tungstate, appears to satisfy these prerequisites. Tomograms derived from multiple projections of EM images of the same structure yielded detailed images of single proteins on the surface of influenza A virus. Comparison of these images with published results from other methods served to evaluate this negative stain tomography. Images of surface renderings of the virus are a good fit to images derived from cryomicroscopy, as well as to the shapes of crystallized surface proteins. Thus, negative stain tomography provides realistic and detailed images of individual molecules in their normal setting on the surface of influenza A virus.
We report on how it is possible to flash-freeze, stain and image at very high magnifications microtubules stabilized with Taxol, using high electron doses, without recording modifications during illumination. This staining mechanism is used since a few years to consistently result in well-defined microtubules [1][2] for tomography experiments. Tentatively, the unexpected dose resistance after elimination of unbound water from the grids, by sublimation before irradiation, may reflect changes in absorption of the materials in the sample at wavelengths larger than visible light. Such radiation would result from parasitic scattering of electrons from the heavy metals in these samples. Diameter of resulting microtubules measured between 25 and 32 nm. High electron dose is reflected in the high definition of these images, acquired with Poisson counting statistics. With such definition, some deformations of microtubule, possibly induced by interaction with stain or substrate, appear. As well, many irregularities on the surface of these microtubules are also present. Because of the high purity of this sample, the large constructs found on the surface of supporting film may derive from an unexpected association of tubulin, possibly involving material from the substrate. These large aggregates bind the stain. Very small-size (diameter ~20Å) globular stained aggregates were found in a control experiment using bovine-serumalbumin (BSA) instead of tubulin. BSA is known to spontaneously aggregate. 11µl of solution of Taxol stabilized microtubules from pure (>95%) rat brain tubulin was deposited using a calibrated pipette on a holey or C-only 200-mesh copper grid (EMS), while it was held by forceps for ~1 minute. Solution resulted from 8µl of microtubules mixed to 3µl of colloidal gold (3nm) on grid. After wicking gently excess, 6µl of uranyl acetate (1% m/v) was deposited for ~30 seconds on grid held by forceps. Then two 8µl drops of methyl-amine tungstate (NanoW, Nanoprobes), diluted 1:50, were used. The first drop was in contact with the grid ~3 s while the second one ~30 s. As the second drop of diluted NanoW was deposited, the grid was quickly mounted in a Leica plunge freezer, blotted on the side, and rapidly (< 3 s) immersed in liquid ethane. Samples were stored for 1-3 days in liquid nitrogen (LN) before use. For experiments, grids were inserted in a Gatan cryo-holder (Model 626) loaded with LN. After insertion in the Jeol 1400 TEM, temperature was raised 5ºC/min to -102ºC and then 1ºC/min to -92ºC. Sample was kept for ~1.2 hours at -92ºC before switching on electron beam at 120 keV. Vacuum was optimal when beam illuminated the grid. Imaging operations were complicated by the fact that sample was not flat because of membranaceous stained material. Therefore, the accessible angular range for tomography measurement was limited. Tomogram in Figure 3 was measured over +55º and -52º using a 1º angular step and 5,000 e -/nm 2 per image. Total dose was ~10 6 e -/nm 2 ; beam was not blanked during motions. Defocus was set in S...
Coherent dynamic x-ray scattering has been used to study the thermally excited layer fluctuations in freely suspended smectic films of the compound 4O.8. Using 8-keV x rays and films with a thickness around 0.3 &mgr;m we resolve relaxation times down to a few &mgr;s. A combination of damped and oscillatory behavior is observed for the layer undulations, which can be attributed to inertial effects. These are due to the surface contribution to the free energy which cannot be disregarded for thin films.
Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a major component of postsynaptic densities (PSDs) involved in synaptic regulation. It has been previously shown that upon activity CaMKII from the spine reversibly aggregates at the cytoplasmic surfaces of PSDs, where it encounters various targets for phosphorylation. Targets for CaMKII are also present within the PSD, but there has been no reliable method to pinpoint whether, or where, CaMKII is located inside the PSD. Here we show that CaMKII can be mapped molecule-by-molecule within isolated PSDs using negative stain electron microscopy tomography. CaMKII molecules found in the core of the PSD may represent a pool distinct from the CaMKII residing at the cytoplasmic surface.
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