1200exii electron microscope

The 26S proteasomes were co-purified with polyubiquitin conjugates on ^GstUBD beads followed by incubation at 37°C for 7 minutes, centrifugation at 12,000 rpm/min for 1 minute and immediate processing of the collected supernatants for EM. Briefly, undiluted 5 µl aliquots were applied to plasma-discharge treated carbon-coated support grids, adsorbed for approximately 30 sec at room temperature, washed with water, and stained with 2% uranyl acetate (unadjusted pH) for approximately 30 sec before blotting and drying. Samples were examined with a JEOL 1200EXII electron microscope, using magnifications of 40,000 or 60,000 to record images on Kodak SO-163 film at either high-dose or minimal-dose conditions. Films displaying several proteasomes per field were scanned using an Epson Perfection V500 Photo flatbed scanner, using resolutions of 846 or 1270 dpi to yield a pixel size of 5Å. All particles recognized as 20S end and side views, and 26S side views, as well as rings and circles of reproducible dimensions, were selected from these scanned images with the EMAN [62] (link) routine Boxer. After eliminating particles that were recognizable but poorly stained, the selected particles were subjected to multiple rounds of reference-free alignment and averaged using SPIDER [63] .

Samples of hArc under different conditions (water; 20 mM sodium Hepes, pH 7.0; 20 mM sodium Hepes, pH 7.0, with 125 mM KCl) were applied to glow-discharged carbon grids at RT for 3 min, washed and then stained for 2 min with 2% (w/v) uranyl acetate. Micrographs were recorded on a JEOL 1200EX-II electron microscope operated at 100 kV on Kodak SO-163 film.