Negative-stain EM of 3D11 IgG-PbCSP complex

To obtain soluble complexes of 3D11-IgG-PbCSP for NS analysis, 8.4 µg of PbCSP was incubated overnight with 20x molar excess of 3D11 IgG. After removal of aggregates via centrifugation, 3D11 IgG-PbCSP complexes were purified on a Superose 6 Increase 10/300 GL column (GE Healthcare). Fractions containing complexes of 3D11 IgG-PbCSP were pooled and concentrated, and subsequently deposited at approximately 50 µg/mL onto homemade carbon grids and stained with 2% uranyl formate. Data were collected with a FEI Tecnai T20 electron microscope operating at 200 kV, and acquired with an Orius charge-coupled device (CCD) camera (Gatan Inc) at a calibrated 34,483X magnification, resulting in a pixel size of 2.71 Å. Particle picking, extraction and three rounds of 2D classification with 50 classes allowed were performed with cryoSPARC v2 (Punjani et al., 2017).