Search PubMed⌕ Search

Biomedical subjects

J Dubochet

Publications and source records attributed to J Dubochet.

At least 73 records · Page 4Linked to original sources
PubMed1978

Nucleosome arcs and helices.

Crystals and other regular arrangements of nucleosome cores have been obtained and analyzed in the electron microscope. Two types of regular structures have been studied in detail, the nucleosome arcs and cylinders. The latter are composed of concentric cylindrical layers of intertwined right-handed helices of nucleosome cores. These studies lead to the following conclusions and concepts. The overall structure of the nucleosome core is a short, wedge-shaped cylinder measuring about 110 by 110 by 60 angstroms. Nucleosome cores interact primarily between top and bottom planes. Nucleosome cores exhibit large conformational variability. A pivot allowing two degrees of rotational freedom is postulated in the region of the 70th base pair to account for this property of the nucleosome.

Animals↗
PubMed1976

A simple method to measure resolution on micrographs.

A method for rapid measurement of the resolution of a microscope by finding the smallest distance between pairs of points that appear on two successive micrographs is described. The method based on the visualization of moire pattern gives the same result as other tests with a precision sufficient for routine checks. Neither a special instrument nor time consuming training are required.

Microscopy↗
PubMedDate not supplied

DNA at the entry-exit of the nucleosome observed by cryoelectron microscopy.

Whereas the DNA path inside the nucleosome is well established, it is essentially unknown in the "entry-exit" region, a missing piece in our understanding of the chromatin fiber's folding. The three-dimensional structure of "linker" DNA was investigated here on single nucleosomes reconstituted without H1 on a 256-bp DNA fragment. Cryoelectron microscopy and three-dimensional reconstruction of the DNA axis reveal these nucleosomes as 1.61 +/- 0.15 left-handed superhelical turn particles whose DNA arms bend away from the core particle, thus preventing the occurrence of a crossing in the entry-exit region.

Animals↗
PubMedDate not supplied

The flying cylinder: a new algorithm for filament recognition in noisy stereo images.

We present a new method of automatic 3D filament representation which uses stereo micrographs to reconstruct three-dimensional trajectories of filament-like objects as DNA molecules. The method deals with low contrasted and noisy images, as obtained from cryovitrified samples by means of electron microscopy. The three-dimensional information is extracted from skeletizing simultaneously both images of a given stereo-pair, instead of processing them separately. The main advantages of the technique are reproducibility and speed, compared to the reconstruction done by manual registration, i.e., clicking on the stereo micrographs.

Computer Graphics↗
PubMedDate not supplied

Cryo-negative staining.

A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thinner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry negative staining. Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast. Examples of cryo-negative staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and the E. coli chaperone GroEL. Densitometric quantitation of the mass-density of cryo-negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen. Determination of the image resolution from cryo-negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca. 10 A and 11.5 A, respectively. For cryo-negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3 A). The electron diffraction resolution is reduced to ca. 10 A when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope. Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV. These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica. Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted. A reconstruction from a subset of undistorted particles produced the characteristic T = 3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography. The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-negative staining. However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule. This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed.

Animals↗