Titre
Cryo-negative staining
Type
article
Institution
UNIL/CHUV/Unisanté + institutions partenaires
Auteur(s)
Adrian, M.
Auteure/Auteur
Dubochet, J.
Auteure/Auteur
Fuller, S. D.
Auteure/Auteur
Harris, J. R.
Auteure/Auteur
Liens vers les personnes
Liens vers les unités
ISSN
0968-4328
Statut éditorial
Publié
Date de publication
1998-06
Volume
29
Numéro
2-3
Première page
145
Dernière page/numéro d’article
60
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Apr-Jun
Research Support, Non-U.S. Gov't --- Old month value: Apr-Jun
Résumé
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.
Sujets
PID Serval
serval:BIB_6F1C626F3EC9
PMID
Date de création
2008-01-24T09:25:31.345Z
Date de création dans IRIS
2025-05-20T22:29:09Z