Temcam f416 cmos

Fab/IgG-SOSIP complexes were made by incubating Fabs or IgGs with AMC009 SOSIP trimers with a 4-fold and 2-fold molar excess of antibody, respectively, for 30 minutes at RT. The Fab/IgG-SOSIP complexes were loaded onto glow-discharged, carbon-coated Cu400 EM grids at a concentration of 30 ng/μl in TBS for 10 seconds. The grids were blotted to remove excess sample and the Fab/IgG-SOSIP complexes were then stained with 2% (w/v) uranyl formate. Grids were immediately blotted and a second stain with 2% (w/v) uranyl formate was applied for 30 seconds, followed by a final blot to remove excess stain. A Tecnai Spirit T12 (FEI) (120kV, 52,000x magnification) equipped with an Eagle 4K CCD (FEI/Thermo Fisher) or Tecnai T20 (FEI) (200kV, 62,000x magnification) equipped with a TemCam F416 CMOS (TVIPS)was used to image the grids. Image collection was performed using Leginon and data processing was carried out as previously described [60 (link),61 (link)]. 2D classification and 3D sorting was performed with Relion v3.0 [62 (link)], and UCSF Chimera [63 (link)] and Segger [64 (link)] were used to visualize and segment the EM maps, respectively.

Cryo-grids were made as reported previously^{24 (link)}. The grid screening process of NaK was performed using an FEI Technai F20 field-emission TEM as before¹⁷ . Continuous rotation MicroED data were collected^{26 (link)} with a TVIPS TemCam-F416 CMOS camera at the rolling-shutter mode as a movie. Each frame in the movie was recorded as the campustage was rotated at 0.19° s⁻¹ during 4 s exposures. Image frames were converted to SMV format for subsequent data processing^{27 (link)}.

Fabs or IgGs were complexed with SOSIP trimers for 30 min at RT with a 4-fold and 2-fold molar excess of Fab and IgG, respectively. Fab/IgG-SOSIP complexes were diluted to 30 ng/µl in TBS and loaded onto glow-discharged, carbon-coated Cu400 EM grids for 10 s followed by blotting to remove excess sample. The Fab/IgG-SOSIP complexes were then stained with 2% (w/v) uranyl formate and immediately blotted, followed by a second stain with 2% (w/v) uranyl formate for 30 s before blotting to remove excess stain. Image collection was performed on a Tecnai Spirit T12 (FEI) (120 kV, ×52,000 magnification) equipped with an Eagle 4 K CCD (FEI/Thermo Fisher) camera or Tecnai T20 (FEI) (200 kV, ×62,000 magnification) equipped with a TemCam F416 CMOS (TVIPS) camera using Leginon. Data processing was performed as follows^{57 (link),58 (link)}. 2D classification and 3D sorting was performed using Relion v3.0⁵⁹ and EM maps were visualized and segmented using UCSF Chimera^{60 (link)} and Segger^{61 (link)}, respectively.