Cryo–electron tomography

Purified influenza virus in HBS (pH 7.50) buffer was rapidly diluted 1:1 with HBS acidification buffer (150 mM NaCl, 10 mM Hepes, 80 mM citrate, and 0.02% NaN3) to a final pH of 5.10 at 37°C and incubated for 1 min. Bovine serum albumin gold beads (10 nm) were then added to sample. C-flat grids (2/2 200 mesh) were glow-discharged for 30sec at 25mA. Using a Vitrobot Mark IV (FEI Co.), 3 μl of sample was applied to the C-flat grids, blotted for 3.5seconds, and plunge-frozen in liquid ethane. Frozen grids were imaged using a 300kV Titan Krios with a Gatan K2 direct electron detector in counting mode. Tilt series were collected using Leginon from −48° to +48° with a 3° step size at a magnification of ×53,000, which corresponds to 2.58 Å per pixel. The total dose per tilt series was ~64 e/Å2. Thirty tilt series were collected and further processed.

Tilt series image frames were corrected for electron beam–induced motion using motioncor2. The images were then ordered going from negative tilt angle to positive tilt angle to create an image stack. These were then processed for tomogram generation using batch tomography in IMOD using standard procedures. Tilt series images were aligned using the gold bead fiducial markers based on routine protocol in the IMOD website. The aligned image stack was then binned twice and reconstructed using weighted back-projection to give 3D tomograms. The final tomograms were rotated around x-axis in IMOD, binned, and low pass–filtered for visualization using IMOD and ImageJ softwares.