(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.

(a) Schematic drawing of the recruitment of target complexes to the Affinity Grid by the His-tagged protein A/antibody adaptor system. (b–d) Representative images and class averages (insets) of negatively stained 60S ribosomal subunits recruited to the Affinity Grid by antibodies against Flag tag (b), Myc tag (c) and rpl26 (d). Scale bar is 60 nm and the side length of the insets is 43 nm. A construct of rpl3 containing an N-terminal tandem Flag-Myc tag was transfected into HEK-293T cells as described in 6. Affinity Grids were prepared according to 7. 3-μl aliquots first of His-tagged protein A (0.1 mg/ml) and then the respective IgG antibody (0.1 mg/ml) were applied to an Affinity Grid for 1 minute each. The excess solution was removed and a 3-μl aliquot of the HEK-293T cell extract was applied. After blotting, the sample was negatively stained according to 6. Specimens were examined using an FEI Tecnai 12 electron microscope (FEI, Hillsboro, OR) equipped with a LaB₆ filament and operated at an acceleration voltage of 120 kV. Images were recorded on imaging plates under low-dose conditions at a nominal magnification of 67,000× and a defocus value of about −1.5 μm. Imaging plates were read out with a DITABIS Micron scanner (DITABIS Digital Biomedical Imaging System AG, Pforzheim, Germany) according to 23. The digitized images were binned over 2 × 2 pixels for a final sampling of 4.5 Å/pixel at the specimen level.

Different views of the final density map (white translucent surface) of human RNAP II filtered to 25 Å resolution calculated from 22,704 particles, into which the crystal structure of yeast RNAP II was placed (Rpb4/7 in red, other subunits in yellow, nucleic acid in blue; PDB entry 1Y77 19). Label “1” indicates the region of the density map into which the atomic model of Rpb4/7 was moved. Label “2” indicates density not accounted for by the crystal structure, which may represent the C-terminal domain of Rpb1 and/or part of the antibody against Rpb1 used to recruit RNAP II to the Affinity Grid. Label “3” indicates the exit channel for the newly synthesized RNA. Scale bar is 5 nm.