Structural Characterization of Antibody-Antigen Complexes

The antibody-antigen complexes at approximately 8 μg/mL were applied to glow-discharged, carbon-coated copper grids. The grids were washed and stained with 0.75% (wt/vol) uranyl formate for 30 sec. The specimens were imaged with a FEI Tecnai F20 electron microscope operated at 120 kV. Micrographs were recorded with a Gatan UltraScan 1000 CCD at 1.21 Å/pixel and at an average defocus of 1.3 μm. The images were corrected for contrast transfer function, and determined the defocus value using CTFfind343 (link). The projections of the particles were boxed using DoG picker44 (link) in Appion45 (link), and classified to 2D classes at 3.63 Å/pixel using the software package ISAC46 (link) and Relion47 (link). The 3D initial models were generated by EMAN2 ab initial common line method48 (link). The initial models were refined with approximately 60,000 particles using Relion47 (link) to 23 Å resolution with gold standard FSC = 0.5 cutoff. Crystal structures of Fab and HER2 ECD (PDB 3wsq) were fit to the 3D model to map the binding region using UCSF chimera49 (link).