Scanning transmission electron microscopy of biological macromolecules

Tichelaar, Willem; Oostergetel, Gert T.; Haker, J.; Heel, M.G. van; Bruggen, E.F.J. Van

doi:10.1016/0304-3991(80)90008-x

Cited by 8 publications

(5 citation statements)

References 16 publications

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“…Uranyl acetate ( l % , wlv) was used for negative staining of the proteins. Scanning transmission electron microscopy was carried out with a Philips-EM400-T equipped with a field emission gun in the cold tip mode and a STEM attachment [20]. The ratio signal of dark field over bright field was used.…”

Section: Electron Microscopymentioning

confidence: 99%

Cyclic tetramolecular complexes of monoclonal antibodies: A new type of cross‐linking reagent

et al. 1986

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A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies, when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1, yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody, stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens, and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine.

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Section: Electron Microscopymentioning

confidence: 99%

Cyclic tetramolecular complexes of monoclonal antibodies: A new type of cross‐linking reagent

et al. 1986

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“…This technique, more than other negative staining techniques, reveals the Y-shape of IgG [28]; physical forces negatively stained areas for reasons given earlier [36], and predominantly footprints of molecules are visualized. Slight embedding increases the chance of deformation of the molecules, but we did not measure a size difference.…”

Section: Discussionmentioning

confidence: 99%

“…Scanning transmission electron microscopy was carried out using a Philips EM 400 equipped with a field emission gun and a STEM attachment [36]. Initially the dark-field signal was used with the field-emission gun in the cold-tip mode.…”

Section: Methodsmentioning

confidence: 99%

The quaternary structure of Escherichia coli RNA polymerase studied with (scanning) transmission (immuno)electron microscopy

Tichelaar¹,

Schutter²,

Arnberg³

et al. 1983

Eur J Biochem

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A model for the quaternary structure of Escherichiu coli RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) is presented. It is based on results from classification of profiles of enzyme molecules, and from application of immuno electron microscopy.Classification of molecules, prepared with the single carbon layer technique, was first achieved for images recorded in dark field with the scanning transmission electron microscope and later on for images recorded in brightfield transmission electron microscopy. It results in five approximately equally sized groups, containing about 80 "/, of the core enzyme profiles. Holoenzyme profiles can be grouped into the same classes, and have approximately the same dimensions (9nm x 16nm). Based on the shapes and sizes of the classified profiles, a tentative model for core enzyme has been constructed.Correlation of shadow projections of this model, with the distributions of attachment sites of antibodies against CI,/I$' and CT over thc profiles, has led to models for core and holoenzyme in which the subunits are localized.The model is compared with literature data on the quaternary structure of RNA polymerase.Eschuiclziu coli RNA polymerase (nucleosidetriphosphate : RNA nucleotidyl transferase) catalyses RNA synthesis. Functional aspects of the enzyme have been extensively studied; for reviews, see 18, 91. Various biochemical and physical techniques have been used to study the quaternary structure of RNA polymerase; for a review, see [lo].In cross-linking studies, /I-/? was found to be the main product for both core and holoenzyme [ll, 121. Using crosslinkers of different lengths, Hillel and Wu [12] found distances of less than 1.2nm between subunits. They presented a model consisting of spherical subunits in which / I and p' form a tetrahedron-like structure together with the M subunits, which constitute a dimer; in holoenzyme the CT subunit interacts with fl and /Y, and with one CI subunit. Partial proteolysis studies [13 -151 support this model. Stender [I61 compared the accessibility of free subunits with their accessibility in situ, using antibodies raised against isolated subunits. He found the CI subunit in situ to be much less accessible than in the isolated state; for b and /j' this difference is much smaller.On the basis of small-angle neutron scattering data, Stockel et al. [I71 presented a model in which the core enzyme is an extended triangle of elongated C I~, /I, and /Y subunits, of which the latter two are curved. They arrived at an axial ratio of 6.1Abbreviurions. TEM, transmission electron microscope/y ; STEM, Enzyme. Escherichia coli RNA polymerase (EC 2.7.7.6).scanning transmission electron microscope/y. for C I~ and /Y, and of 5.6 for 1. The distances between the centres of mass of these subunits range from 7.0 nm to 8.2 nm. In the holoenzyme the CT subunit, for which they derived the shape of an elongated disc, nestles up to the core enzyme in a spacefilling manner; the centre-of-subunit structure forms a tetrahedron 1181.Us...

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“…It is possible to collect both the elastically and the inelastically scattered electrons simultaneously with an energy analyser and, if a fine electron beam is scanned over the specimen, then a scanning transmission electron micrograph displaying different properties of the specimen can be obtained (e.g. Tichelaar et al, 1980;Crewe, 1983;Muller et al, 2008). Alternatively, conventional transmission electron microscopes to which an energy filter has been added can be used to select out a certain energy band of the electrons from the image (e.g.…”

Section: Radiation Damagementioning

confidence: 99%

Electron Cryomicroscopy of Biological Macromolecules

Henderson

Baker

2003

Burger's Medicinal Chemistry and Drug Discovery

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Electron lenses are greatly superior to X‐ray lenses and can be used to produce a magnified image of an object as easily as a diffraction pattern. This then allows the electron microscope to be switched back and forth instantly between imaging and diffraction modes so that the image of a single molecule at any magnification can be obtained as conveniently as the electron diffraction pattern of a thin crystal. This chapter presents a comprehensive survey of the electron microscopy of biological molecules.

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Scanning transmission electron microscopy of biological macromolecules

Cited by 8 publications

References 16 publications

Cyclic tetramolecular complexes of monoclonal antibodies: A new type of cross‐linking reagent

Cyclic tetramolecular complexes of monoclonal antibodies: A new type of cross‐linking reagent

The quaternary structure of Escherichia coli RNA polymerase studied with (scanning) transmission (immuno)electron microscopy

Electron Cryomicroscopy of Biological Macromolecules

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