Trimer was complexed with Moo1 Fab and RM20A3 base Fab at 3x molar excess Fab:trimer overnight at room temperature. The protein complex was then diluted to 0.02 mg/ml in 1x Tris-buffered saline, and 3 μL were applied to a 400 mesh Cu grid, blotted with filter paper and stained with 2% (w/v) uranyl formate. Micrographs were collected on a 120 keV FEI Tecnai TF20 microscope with a TVIPS TemCam F416 CMOS camera (1.68 Å/pixel; 62,000x magnification) using Leginon[52 (link)]. Particles were picked using DoGPicker and 2D and 3D classifications and 3D refinement was performed using Relion 3.0 [53 (link), 54 (link)]. The final map was segmented in UCSF Chimera to generate figures, and deposited to the Electron Microscopy Data Bank (EMDB) [55 (link)].
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Trimer-Fab Complex Structural Analysis
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Other organizations : Scripps Research Institute, Kansas State University, Applied Biotechnology Institute, International AIDS Vaccine Initiative
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Variable analysis
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- Trimer was complexed with Moo1 Fab and RM20A3 base Fab at 3x molar excess Fab:trimer overnight at room temperature
- Protein complex was diluted to 0.02 mg/ml in 1x Tris-buffered saline
- 3 μL were applied to a 400 mesh Cu grid, blotted with filter paper and stained with 2% (w/v) uranyl formate
- Micrographs were collected on a 120 keV FEI Tecnai TF20 microscope with a TVIPS TemCam F416 CMOS camera (1.68 Å/pixel; 62,000x magnification) using Leginon
- Particles were picked using DoGPicker and 2D and 3D classifications and 3D refinement was performed using Relion 3.0
- The final map was segmented in UCSF Chimera to generate figures, and deposited to the Electron Microscopy Data Bank (EMDB)
- Overnight incubation at room temperature
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