文档介绍：Electron Microscopy
of Protein-Nucleic plexes
Enhanced High-Resolution Shadowing
Carla W! Gray
1. Introduction
I describe herein a method in which pretreatment with low concen-
trations of uranyl acetate is used to increase the structural rigidity of
protein-nucleic plexes, thereby substantially enhancing the
information content of images obtained by high-resolution shadow-
casting with tungsten, The visualization of three-dimensional objects
by heavy-metal shadowcasting is along-established technique in elec-
tron microscopy (I), and it has long been realized that the highest
resolution could be achieved by using carbon-metal mixtures or high-
melting-point metals for the evaporation (2,3). Double-stranded DNAs,
2 nm in diameter, are readily visualized by high-resolution shadowing
with tungsten (4).
Protein-plexes formed in vitro by mixing the M 13 or fd
gene 5 single-stranded DNA binding protein* with single-stranded
DNA were shown by negative staining in an early study (5) to be
apparently helical, with an estimated helix diameter of 10 nm and an
approx -7 nm distance between helical turns. Such dimensionally
*The Ml3 and fd gene 5 proteins are single-stranded DNA binding proteins of identical
amino acid sequence encoded by the closely related Ml3 and fd strains of filamentous bacte-
rial viruses.
From: Methods n Molecular Btology, Vol. 22 Mmoscopy, Opttcal Spectroscopy,
and Macroscopic Technques Edlted by C. Jones, B. Mulloy, and A H. Thomas
Copynght 01994 Humana Press Inc , Totowa, NJ
1
2 Gray
Fig. 1. Complex of fd gene 5 protein with viral DNA, formed by mixing the
protein and DNA in vitro. plex was adsorbed from ammonium acetate
(pH ) onto a glow-discharge-activated carbon, dehydrated by passage through
graded ethanol solutions before air-drying (6), and rotary-shadowed with tungsten.
The plexes rinsed with water rather than ethanol have essentially the
same appearance