Computational protocol: Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

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Protocol publication

[…] BG505 SOSIP.664 + NC-Cow1 complexes were placed on glow-discharged carbon coated copper mesh grids and stained with 2% uranyl formate. Grids were screened for appropriate stain thickness and particle distribution using an FEI Morgagni (80keV) electron microscope. The final data set was collected on an FEI Tecnai Spirit T12 (120keV) electron microscope with a Tietz TVIPS CMOS (4K by 4K) camera controlled with Leginon automated imaging software. Images were collected at 52,000× magnification with a −1.3 μm defocus for a final magnified pixel size of 2.05 Å/pix. Both Automated particle picking performed with DoG-Picker and reference-free 2-D classification with iterative MRA-MSA were executed through the Appion database. For 3D analysis, micrographs were first CTF estimated with GCTF then particles were extracted, phase-flipped, and subjected to reference-based 3D classification and refinement in Relion version 2.0. The final 3D reconstruction contained ~3.5k particles out of the initial ~13.5k that went into 3D classification. UCSF Chimera was used to generate figures. […]

Pipeline specifications

Software tools Leginon, DoG Picker, Appion, RELION, UCSF Chimera
Application cryo-EM
Organisms Bos taurus, Homo sapiens, Oryctolagus cuniculus, Macaca fascicularis
Diseases HIV Infections
Chemicals Amino Acids