ABSTRACT
A computer search of the recent literature will reveal that both negative staining and cryoelectron microscopy of unstained vitrified specimens continue to be widely used (e.g. Marr et al., 1996; Zhao et al., 1995). The marked decline in the use of negative staining, predicted by some 10 or more years ago, in reality has not happened and the technique continues to be useful and even expand in some areas. We have proposed (Harris and Horne, 1994) that with further technical development negative staining should continue to be valuable for macromolecular studies, and it is now clear that improved resolutions also can be achieved. The increasing availability of electron microscopes with cryospecimen holders means that the study of negatively stained specimens prepared in the presence of carbohydrate (trehalose or glucose) can benefit from cooling to temperatures below −170°C (Orlova et al., unpublished observations). Under these conditions (with minimal adsorption of molecules to the carbon film) the achievable resolution from negative staining can be better than 15 Å within 3-D reconstructions, as shown by Dube et al. (1995) for the earthworm, Lumbricus terrestris, haemoglobin ( Figure 9.1 ). The amplitude contrast is higher within negatively stained specimens than within unstained specimens in vitreous ice, so images can be recorded closer to Scherzer focus and within the plateau of the contrast transfer function, which may ultimately yield resolutions better than 10 Å. Indeed, it should be borne in mind that specimens embedded within a dried layer of negative stain also appear to be considerably less labile within the electron beam than unstained specimens in vitreous ice. However, this may be predominately lower resolution information that is being preserved by the negative stain, because of the inherent limitations of embedding in a high contrast medium. The generally held belief that negative staining will reveal only the surface contour of a protein is undoubtedly correct (Henderson, 1995). Providing the negative staining ion can penetrate the water-space within and around an intricately folded polypeptide chain then meaningful detail of quaternary structure will be obtained. Once the negative stain is unable to enter a very small water-filled surface channel or totally enclosed compartment within a protein, the technique will clearly have come to its limitation. The definition of individual domains of a protein can often provide valuable information; this should now be more routinely achievable from negative staining at resolutions in the range 10–15 Å. 3-D reconstruction of earthworm haemoglobin.
Earthworm haemoglobin: 3-D reconstruction (imagic-5) at 15 Å from a low temperature study of ammonium molydate-glucose specimens (Dube et al., 1995). This series of tilted molecules (a continuous stereo sequence) can be observed as a magic-eye stereo-array, with a little practice (cf. Figure 5.33 ). Reconstruction courtesy of Marin van Heel and Prakash Dube.
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