Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate

Abstract

The mechanism of contrast enhancement of protein molecules by negative staining with uranyl acetate has been investigated by analysing electron micrographs of microcrystals of the human immunoglobulin Dob. Digitally filtered micrographs were compared systematically with idealized reference images which were constructed computationally, starting from knowledge of the primary sequence and three-dimensional crystal structure of this IgG molecule. By separately modelling negative staining as bulk exclusion of heavy metals, and positive staining as the specific decoration of charged amino acid residues, and then combining these simulated images, we were able to assess quantitatively the amount of positive staining present in micrographs of ostensibly 'negativelfstained' proteins. At a resolution of 2 nm, we find that the experimental images do indeed exhibit predominantly negative staining, the best matches being obtained by simulations which also include a minor contribution (1040%) of positive staining. We have also compared two independent measures for the significant resolution present in images of periodic biological specimens : (i) the outermost visible orders of optical diffraction patterns, and (ii) the band-limited resolutions of the idealized simulations when they most closely match the experimental images. These criteria observe close correspondence, thus vindicating the traditional practice of inferring resolution from the optical diffraction spectra of indirectly represented (stained) objects.

Visualization of bionanostructures using transmission electron microscopical techniques

“…Viruses are built up by an RNA or DNA genome surrounded by a protective protein capsid. The majority of capsids show an icosahedral or helical symmetry (Steven et al,1997); rarely, conical (e.g., human immunodeficiency virus) or complex shapes (e.g., pox virus) are observed (Cyrklaff et al,2005; Ganser‐Pornillos et al,2008). In addition, some viruses also consist of a membrane envelope with embedded proteins.…”

“…However, the degree of structural stability and conformance with the symmetry differs among the individual viruses (Steven et al,1997). Cryo‐EM of those viruses that obey the symmetry constraints can provide near‐atomic details into the architectures of the viral particles (Sachse et al,2007; Yu et al,2008; Zhang et al,2008b,2010b).…”

Tau protein becomes long and stiff upon phosphorylation: correlation between paracrystalline structure and degree of phosphorylation.

“…Moreover, there is little difference between negative and positive staining by uranyl acetate. This stain has a strong affinity for charged groups (positive and negative) so that even the images of negatively stained structures contain an appreciable contribution from positive staining (compare studies with immunoglobulins, Steven and Navia, 1982;or collagen, Tzaphlidou et al, 1982). Since tau has a high content of charged residues we assume that the images of uranyl acetate-stained paracrystals largely reflect the charge distribution.…”

“…3 the minus end is at the bottom, the plus end is at the top. We will denote the region containing groups (Steven and Navia, 1982) this suggests that this stain represents largely the charge distribution, even when used as a negative stain.…”

Introduction to the computer image processing of electron micrographs of two‐dimensionally ordered biological structures

“…However, globular molecules may also have clusters of charged residues on their surface, and this can give rise to an artefactually high local concentration of charge that will attract stain and so be interpreted as a hole in the structure rather than its surface. Steven and Navia (1982) present an illustrative analysis of the degree of positive staining in immunoglobulin crystals. These problems can sometimes be resolved by using a range of different stains and by shadowing, but the possibility of positive staining should always be born in mind when interpreting images at resolutions higher than about 3 nm.…”