Extended Data Fig. 7: Characterization of LALA-PG mutant mAbs and negative stain electron microscopy of Spike-Fab complexes.
a, Characterization of the LALA-PG mutant forms of the NTD mAbs WRAIR-2039, 2025 and 2004 (in blue) and RBD mAbs WRAIR-2173 and 2123 (in red) in cell surface S binding (left), ADCD (middle) and ADNP (right) assays. Non-significant differences in binding to cell surface S was observed between WT and mutants in a Wilcoxon matched-pairs signed rank test, P = 0.3125. LALA-PG mutants did not show any activity in the ADCD and ADNP assays. b, Neutralization curves of WT and LALA-PG versions of WRAIR-2039 (NTD) and -2123 (RBD) mAbs obtained in the pSV assay. Shown are mean ± SD from at least 2 independent experiments. IC50 (µg ml-1) are indicated in parentheses in the legend. c, EM analysis of WRAIR-2173 and WRAIR-2025 Fabs in complex with SARS-CoV-2 spike (S-2P) trimer. Left panel: Raw image (top) and two-dimensional class particle averages (bottom, 5 averages shown). The black bars represent 500 Å. Middle panel: Gold-standard FSC curves for the EM 3D reconstruction. Right panel: Negative-stain 3D reconstruction of SARS-CoV-2 spike and Fab complex. The structural model of the SARS-CoV-2 trimer (PDB 6X2B) in complex with WRAIR-2173 and WRAIR-2025 Fabs is shown in ribbon representation while negative-stain electron density map is shown as a gray transparent surface (also shown in Fig. 5b). A featureless and unbiased (lowpass filter: 100Å) 3D model of SARS-CoV-2 spike trimer (PDB: 6VXX) and 12,574 particles were used to perform the 3D reconstruction from a single experiment. d, EM analysis of WRAIR-2025 Fab in complex with SARS-CoV-2 spike (S-2P) trimer. Left panel: Raw image (top) and two-dimensional class averages (bottom, 5 averages shown) of Fab-Spike particles. The black scale bars represent 500 Å. Middle panel: Gold-standard FSC curves for the EM 3D reconstruction. Right panel: Negative-stain 3D reconstruction of SARS-CoV-2 Spike and Fab complex. The structural model of the SARS-CoV-2 trimer (PDB 6VXX) and WRAIR-2025 Fab is shown in ribbon representation while negative-stain electron density map is shown as a gray transparent surface. A feature less and unbiased (lowpass filter: 100Å) 3D model of SARS-CoV-2 spike trimer (PDB: 6VXX) and 3,364 particles were used to perform the 3D reconstruction from a single experiment.
