Digital acquisition and processing of electron micrographs using a scanning transmission electron microscope

Engel, Andréas; Christen, Fabienne; Michel, B.

doi:10.1016/0304-3991(81)90022-x

Cited by 41 publications

(8 citation statements)

References 7 publications

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“…5), and therefore raise the question about the existence of systematic errors. To this end, we can exclude preferential adsorption of salts, detergent, or small polypeptides to the protein structures, since the mass-per-length measured for tobacco mosaic virus coincided with the theoretical value within 10% (see Engel et al, 1981). In addition, our estimate for the contribution of the embracing nuclear envelope to the mass of intact membrane-bound NPCs (see Materials and Methods) appears to be correct, since their corrected mass (i.e., 124 + 11 MD) closely matches that of intact but membrane-detached NPCs (i.e., 125 + 12 MD).…”

Section: Discussionmentioning

confidence: 99%

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Reichelt

Holzenburg

Buhle

et al. 1990

The Journal of Cell Biology

479

298

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Abstract. Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings" "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 + 11 MD; intact NPC without plug, 112 + 11 MD; heavy ring, 32 + 5 MD; light ring, 21 ± 4 MD; plug-spoke complex, 66 ± 8 MD; and spoke complex, 52 ± 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

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Section: Discussionmentioning

confidence: 99%

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Reichelt

Holzenburg

Buhle

et al. 1990

The Journal of Cell Biology

479

298

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“…Mass determination by STEM thus became a powerful tool for the analysis of macromolecular assemblies (Engel, 1978;Wall, 1979). Since then the data acquisition (Engel et al, 1981;Wall et al, 1973), software and specimen preparation for mass determination has been improved and continuously applied to biological macromolecules and assemblies thereof Engel, 1998, 2001;Wall and Simon, 2001;Wall et al, 1998). Indeed, the dedicated STEM has become a valuable tool in biophysical research.…”

Section: Introductionmentioning

confidence: 98%

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

Krzyžánek

Müller

Engel

et al. 2009

Journal of Structural Biology

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“…The mass of the LP complex was computed from integrated scattered intensity inside a circle slightly larger than the LP complex diameter (=350 A). For unknown reasons, the TMV was not an adequate standard (17) in measuring the masses of hook-containing complexes. For these structures, three sets of measurements were made: (i) total scattering intensity of the particle IHC, (ii) hook linear scattering intensity IH/L, and (iii) hook length LH.…”

Section: Methodsmentioning

confidence: 99%

Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

Sosinsky

Francis

DeRosier

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The basal body, a part of the rotary motor of the bacterial flagellum, is a multiprotein assembly that consists of four rings (denoted M, S, P, and L) and an axial rod (denoted R). From analysis of scanning transmission electron microscopy images of hook-basal body preparations isolated from Salmonella typhimurium, we have determined the masses of the basal body and three of its subcomplexes. The mass of the basal body (i.e., the four rings and rod) is 4400 ± 490 kDa (mean ± SD; n = 54). The mass of the LPR subcomplex (i.e., L and P rings and the whole rod) is 2600 ± 380 kDa (n = 55), that of the L and P rings and the distal part of the rod is 2100 ± 320 kDa (n = 25), and the mass of the L and P ring subcomplex is 1700 ± 260 kDa (n = 514). These results, together with the masses of the component proteins, indicate that the rings contain -26 subunits each and that the mass of the rod is consistent with a composition of ""6 copies each of three of the The bacterial flagellum, the organelle responsible for cell motility, is a macromolecular assembly consisting of multiple copies of at least 13 different proteins. The flagellum contains an axial structure running its entire length and a set of ring structures that are localized in the basal body (see Fig. 1 Inset). In Salmonella typhimurium, the components of the axial structure, in cell proximal-to-distal order, are FlgB, FlgC, and FlgF (proximal rod proteins); FlgG (distal rod protein); FIgE (hook protein); FlgK and FlgL (HAP1 and HAP3, the filament-hook junction proteins); FliC (flagellin, the filament protein); and FliD (HAP2, the filament capping protein). The basal body, the part of the flagellum embedded in the cell envelope, contains four rings-M, S, L, and P. The M ring is made from the FliF protein (1), which also makes up the S ring and the cell proximal part of the rod (T. Ueno, K. Oosawa, and S.-I. Aizawa, personal communication). The P ring and the L ring are composed of FlgH and FlgI, respectively (2). An additional protein, FliE, is also a basal body protein, but its location is unknown (3).In an earlier paper, we described the hook-basal body complex (HBB) and its subcomplexes obtained by dissociation in acid (4). Using low-dose electron cryomicroscopy and image-averaging techniques, we computed averages of the HBB and four subcomplexes at -"25 A resolution. In these averaged images, the S, P, and L rings appear cylindrically symmetric, but the M ring shows some evidence of angular periodicity. We were unable to deduce the subunit symmetry of the constituent proteins from these reconstructions or from the individual images of isolated L-P ring structures (LP) lying en face. (6) or 12 (7), which were based on inspection of electron microscope images of negatively stained isolated LP. In addition, freeze-fracture electron micrographs ofEscherichia coli cell membranes contain rings of 11 or 12 intramembranous particles, which are thought to be the MotA and MotB proteins (8). However, it is not known whether these rings interact with the M ring...

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Digital acquisition and processing of electron micrographs using a scanning transmission electron microscope

Cited by 41 publications

References 7 publications

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

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