Microscopy, Optical Spectroscopy, and Macroscopic Techniques

Electron Microscopy of Protein-Nucleic Acid Complexes Enhanced High-Resolution

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Carla W! Gray 1. Introduction I describe herein a method in which pretreatment with low concentrations of uranyl acetate is used to increase the structural rigidity of protein-nucleic acid complexes, thereby substantially enhancing the information content of images obtained by high-resolution shadowcasting with tungsten, The visualization of three-dimensional objects by heavy-metal shadowcasting is along-established technique in electron microscopy (I), and it has long been realized that the highest resolution could be achieved by using carbon-metal mixtures or highmelting-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-DNA complexes 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 6.5-7 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 bacterial viruses. From: Methods and Macroscopic

n Molecular Btology, Vol. 22 Mmoscopy, Opttcal Spectroscopy, Technques Edlted by C. Jones, B. Mulloy, and A H. Thomas Copynght 01994 Humana Press Inc , Totowa, NJ

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Fig. 1. Complex of fd gene 5 protein with viral DNA, formed by mixing the protein and DNA in vitro. The complex was adsorbed from O.OlM ammonium acetate (pH 6.9) onto a glow-discharge-activated carbon, dehydrated by passage through graded ethanol solutions before air-drying (6), and rotary-shadowed with tungsten. The same complexes rinsed with water rather than ethanol have essentially the same appearance as shown here, as is also true for fd complexes isolated from infected cells and prepared as described here with drying from water or ethanol. Scale bar = 50 nm.

large structural features should be resolved by tungsten shadowing. It was quite surprising, therefore, to find that complexes of Ml3 viral DNA with the Ml3 gene 5 protein, isolated from virus-infected bacterial cells, did not show the expected three-dimensional structure when shadowed with tungsten (6). This finding led to the conclusion (which was later shown to be incorrect [7,8]) that complexes formed in vivo did not have the same structure as had been shown by negative staining for complexes reconstituted in vitro. Why did tungsten shadowing fail to delineate the helical structure in the case just cited? Poor structural definition is also obtained in my laboratory upon tungsten shadowing of in vivo and in vitro complexes of the fd gene 5 protein with fd viral DNA, using methods similar to those used in (6); an example is shown in Fig. 1. Although the shadowed complex in the figure has roughly the expected overall dimensions (length and width), the individual helical turns cannot be distinguished, and the detailed structure is irregular. A possible reason for the lack of structural definition was revealed by our recent studies

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(8) using uranyl acetate staining and specimen tilting to elucidate the three-dimensional structures of the fd complexes. In those studies, it was found that considerable flattening of the complexes occurs upon adsorption to glow-discharge-activated carbon; calculations indicated that the apparent diameter of 9.5-10.5 nm for the flattened helices should be corrected to a diameter of 7-9 nm before flattening. Flattening of a specimen will of course reduce the resolution of its structural features by shadowcasting, which depends on the elevation of discrete structures above the supporting surface and on their spatial separation from neighboring structures. Moreover, the uranyl acetate stain used in the tilting experiments is itself believed to provide structural support for specimens partially embedded in the stain (9,10). Thus, it seemed likely that even more pronounced flattening of the M 13 or fd protein-DNA complexes could have occurred in the preparations used for shadowcasting, in which the specimens were dried from aqueous or ethanolic solutions in the absence of uranyl acetate.

I therefore set about to see whether it would be possible to develop a technique combining high-resolution tungsten shadowing of nucleoprotein complexes with the use of uranyl acetate stain as a medium for specimen support. Enhanced contrast haspreviously been observed for DNAs stained with uranyl acetate prior to shadowing (11,12). Surprisingly, I found that pretreatment with uranyl acetate provided substantial support and greatly improved structural resolution in shadowed nucleoprotein complexes, even at concentrations below those required for the uranyl salts to be visible as a stain. The specimen contrast in Figs. 2-4 is entirely owing to the tungsten deposits and not to the uranyl acetate, as was determined in tests that are described together with the method. 2. Materials 1. The protein-DNA complexes to be visualized: These must be freshly purified, and free of contaminating proteins, lipids, oils, salts, other nonvolatile components, detergents, and nonaqueous solvents. There

must be no contaminating nucleasesor proteases.We frequently find it necessary to repurify proteins and nucleic acids obtained commercially or from other laboratories. Simple dialysis may suffice to remove lowmol-wt contaminants, or ethanol precipitation (of nucleic acids) or gel ftltratron may be used. It is preferable to mix the DNAs and protems Just prior to mounting them for microscopy; the proteins should not be

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Fig. 2. Complexes of fd gene 5 protein with viral DNA. The complexeswere isolated from infected cells, adsorbedto aglow-discharge-activated carbon, rinsed with water, treated with 0.5% uranyl acetate,and rotary-shadowed with tungsten. The arrows point to individual turns of the nucleoprotein helix that are visible after uranyl acetatepretreatment.Scalebar = 50 nm. present in such excess quantities that they will contribute significantly to the background. About 0.2-2 pm01 of DNA (measured as the concentration of nucleotide phosphates), together with protein added at an appropriate ratio, will be needed in each 50-pL mixture used for adsorption of the protein-DNA complexes to a single specimen grid. 2. Purified water: Water is distilled and then deionized in a “Mini-Q” system (Millipore Corp., Bedford, MA) consisting of one cellulose ester prefilter cartridge, two ion-exchange cartridges, and a 0.22~pm filter, in series. No activated charcoal filter is included in our system, because of a tendency of this filter to release minute charcoal particles. The

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Fig. 3. A complex prepared as for Fig. 2, printed directly from the electronimage negative (left) or from a copy negative having reversed contrast (right). Note the dark shadow around the complex on the right, corresponding to a light halo seen before contrast reversal. The shadow demonstrates that the structure is threedimensional. Scale bar = 50 nm.

Milli-Q system is constructed of highly inert, noneluting materials, and we find that the water (18 megJn/cm as dispensed) can be used for most procedures in electron microscopy. Alternatively, we sometimes use tap distilled water that has been redistilled through a series of two 24-in. borosilicate glass Vigreux columns. 3. Buffers for mixing of the protein and DNA to form complexes: The use of a minimal number of buffer components and a low concentration of salts (