Figure 2. Images of vitrified RNAP II purified onto Affinity Grids using His-tagged protein A and an antibody against Rpb1.

(a) Image of vitrified RNAP II prepared onto an Affinity Grid from HEK-293T cell extract, showing that many of the complexes are associated with nucleic acid strands. (b) Treatment of the cell extract (0.1 ml of ~3 mg/ml protein) with 10 units of RNase A for 30 minutes prior to application to the Affinity Grid did not eliminate the strands. (c) Treatment of the cell extract with 10 units of DNase I for 30 minutes prior to application to the Affinity Grid eliminated the majority of the strands, identifying the strands to be DNA. Vitrified specimens of RNAP II were prepared according to ⁷ and transferred into an FEI F20 electron microscope equipped with a field emission gun using an Oxford cryo-specimen holder, maintaining a temperature of −180°C. Samples were examined at an acceleration voltage of 200 kV and images were recorded on Kodak SO-163 film at a nominal magnification of 50,000× using low-dose procedures and a defocus ranging from −2 to −4 μm. The film was developed and digitized as described ⁶ for a final sampling of 4.2 Å/pixel at the specimen level. Scale bar is 50 nm.