Fcf400 cu 50

All negative-stain imagings in this paper were performed using a Morgagni 268(D) transmission electron microscope S3 microscope (FEI Co.). The microscope is equipped with a tungsten filament operated at 100 Kv high tension, and a Gatan Orius SC200 CCD Detector with a physical pixel size of 7.4 µm. Images well acquired at nominal magnification of 22,000x (2.1 Å/pixel) at room temperature using Leginon (69 (link)). The samples were fixed using conventional negative staining procedures with 0.075% uranyl formate on glow-discharged grids with Formvar carbon film (Electron Microscopy Sciences, FCF-400-Cu-50).
Samples for negative staining of HIV-1 cores with dynein tail were prepared by mixing HIV-1 cores (final concentration of 0.1 nM) with dynein tail (final concentration of 64 nM) in the DLB buffer (final salt 45 mM and 1 mM ATP). The sample was incubated on ice for 30 min and negatively stained without further dilution. The sample of assembled HIV-1 capsid tubes and cones were diluted 10X and 100X, respectively, before negative staining.

Formvar/carbon-coated copper grid (Electron Microscopy Sciences, catalog number FCF400-Cu-50) was glow-discharged and covered with 6 μl of the sample for 1 min before blotting away the sample. The sample was double-stained with 6 μl of 2% (w/v) uranyl formate (Electron Microscopy Sciences, catalog number 22450) for 5 seconds (first stain) and 1 min (second stain), blotting away after each stain. Images were collected using a JEOL JEM-1400 Plus microscope with an acceleration voltage of 80 kV and a bottom-mount charge-coupled device camera (4k by 3k, Advanced Microscopy Technologies).