Computational protocol: Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein

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Protocol publication

[…] Electron microscopy and 2D class averaging were performed by NanoImaging Services, Inc. (San Diego, CA). Samples were prepared on continuous carbon films supported on nitrocellulose-coated 400 mesh copper grids (Ted Pella). A 3 μL drop of purified RSV F protein at a concentration of 2–8 μg/mL was applied to a freshly plasma-cleaned grid for 1 min and blotted to a thin film using filter paper. The sample was washed four times by floating the grid on a droplet of H2O for 1 min followed by staining on a droplet of 3% (w/v) uranyl formate for 1 min. The grid was blotted after each incubation and air-dried. Transmission electron microscopy was performed using an FEI Tecnai T12 electron microscope operating at 120 kV equipped with an FEI Eagle 4k x 4k CCD camera. Images were collected at nominal magnifications of 110,000x (0.10 nm/pixel), 67,000x (0.16 nm/pixel), 52,000x (0.21 nm/pixel) and 21,000x (0.5 nm/pixel) using the automated image acquisition software package Leginon []. Images were acquired at a nominal underfocus of -2 μm to -1 μm (110,000x), -3 μm to -1 μm (67,000x), -4 μm to -2 μm (52,000) and -4 μm (21,000x) and electron doses of approximately 9-39e/Å2.Image processing was performed using the Appion software package []. Contrast transfer functions of the images were corrected using Ace2 []. Individual particles in the 67,000x and 110,000x images were selected using automated picking protocols, followed by several rounds of reference-free alignment and classification based on the XMIPP processing package to sort them into self-similar groups []. […]

Pipeline specifications

Software tools Leginon, Appion, Xmipp
Application cryo-EM
Organisms Respiratory syncytial virus, Homo sapiens, Rous sarcoma virus
Diseases Respiratory Tract Diseases