Structures of small subunit ribosomal RNAsin situ fromEscherichia coli andThermomyces lanuginosus

SummaryThe structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441-444, 1995; Stark et al., Structure, 3, 815-821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.

Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three‐dimensional reconstruction

Beniac

Czarnota

Rutherford

et al. 1997

“…The short‐range order (1 nm resolution) of organic material is already destroyed at doses below 1000 el nm −2 ( Chiu & Jeng, 1982; Lamvik, 1991b). High‐resolution studies of biological material by ESI are therefore difficult, and only feasible if a large number of identical motifs can be averaged ( Beniac & Harauz, 1995). Loss of mass, on the other hand, is critical in ESI, since the irradiation dose must not exceed that limit at which the element of interest would disappear.…”

Section: Introductionmentioning

confidence: 99%

Electron energy‐loss spectroscopy (EELS) of iodine in thyroxine: parameters of radiation‐induced loss of iodine

Haking

Richter

TRÖster

et al. 1998

The loss of iodine during irradiation with electrons was measured in order to determine whether it can be detected by electron spectroscopic imaging (ESI). Since iodine can be bound to organic molecules, it could be a candidate for tracer studies by ESI in biomedical research. A solution of thyroxine spread over a carbon film was used as a test specimen. Thyroxine contains four iodine atoms covalently bound to aromatic rings. For ESI, higher doses have to be used than in conventional electron microscopy. The iodine content was therefore measured after irradiation with doses of up to 3 × 107 el nm−2. The measurements were carried out for different specimen thicknesses, temperatures and dose rates, and it was found that iodine can be detected with ESI if moderate dose rates are used at a temperature of −160 °C.

“…Energy-filtering transmission electron microscopy (EFTEM) has become an established method widely applied to biological questions (Ottensmeyer & Andrew, 1980;De Bruijn et al, 1993;Reimer, 1995). Among these, EFTEM has been used for the selective visualization of phosphorusrich regions such as ribosomes (Korn et al, 1983;Martin et al, 1989;Koenig & Martin, 1996), ribonucleoprotein particles (Bazett-Jones, 1988;Stracke & Martin, 1991), isolated DNA , DNA-protein complexes (Bazett-Jones et al, 1994), kinetochores (Rattner & Bazett-Jones, 1989), Balbiani rings (Olins et al, 1992) and recently for the three-dimensional reconstruction of the RNA backbone of ribosomal subunits (Beniac & Harauz, 1995).…”

Section: Introductionmentioning

confidence: 99%

Detection of low phosphorus contents in neurofilaments of squid axons by Image‐EELS contrast spectroscopy

Door

Richter

Martin

1997

SummaryWe show that Image-EELS is suitable for detecting relatively low phosphorus concentrations in very small axoplasmic structures of squid axons. Imaging plates and a CCD camera were used as electron sensors. From image series spanning a certain energy-loss range EELS (electron energy-loss spectra) were derived by averaging read-outs from many axoplasmic particles (APs). The ratio of these spectra to spectra of the background was plotted, showing the contrast modulation as a function of the energy loss. This new approach is called EELC (electron energy-loss-dependent contrast spectroscopy). A distinct phosphorus signal was found in APs of presynaptic terminals of the squid giant synapse, in the peripheral giant axon and, as controls, in ribosomes. Biochemical experiments supported this result. In neurofilament-enriched pellets a phosphorus signal could be directly detected by serial EELS and in electron spectroscopic micrographs. After dephosphorylation of either the pellets or the extruded axoplasm with alkaline phosphatase, phosphorus signals in electron spectroscopic micrographs were absent or much reduced in size and intensity.With Image-EELS inherent limitations of traditional element detection modes in energy filtering transmission electron microscopy can be overcome. Compared with serial EELS, the selective analysis of small areas with irregular shape is possible with greatly improved signal-to-noise ratio. The identification of the element-peak in Image-EEL spectra directly proves the presence of the element within the region of interest. For small peaks, the visualization is facilitated by the contrast presentation (EELC). However, the background subtraction modes used for elemental mapping in electron spectroscopic imaging are subject to uncertainties when elemental ionization edges like the P L2,3 edge are examined.Imaging plates are very sensitive electron sensors with a wide dynamic range. Unlike photographic emulsions, they allow acquisition of image series covering a large energyloss range without normalization of exposure times, and direct extraction of EEL spectra. Thus, the combination of Image-EELS and imaging plates is proposed as an efficient new tool for analytical electron microscopy.