Crystallography

Fab fragments were generated from full length IgG1 antibodies through cleavage with IgG degrading enzyme E (IGDE) for 16-24 hours at 37°C. Fab fragments were purified using Lambda and Kappa Select columns (Cytiva) per the manufacturer’s protocol. Complexes of SARS-CoV-2 RBD bound to Fab fragments were purified via SEC in 20 mM HEPES pH 7.5, 150 mM NaCl. Crystallization trials were set up with protein concentrations of 8 and 4 mg/ml at the Collaborative Crystallization Centre at Commonwealth Scientific and Industrial Research Organization (CSIROC3, Parkville) at 20°C. Crystals of SARS-CoV-2 RBD- PDI 37 appeared in 0.2 M ammonium sulfate, 20% PEG3350, 0.1 M Tris-chloride pH 8.5 and were harvested with mother liquor containing 25% glycerol. SARS-CoV-2 RBD-PDI 42 crystals were obtained in 10% PEG8000, 0.2 M NaCl, 0.1 M sodium dihydrogen-dipotassium hydrogen phosphate pH 6.2 and were flash frozen in mother liquor containing 30% glycerol. Crystals of SARS-CoV-2 RBD-PDI 210 appeared in 15% PEG6000, 0.1% (w/v) n-Octyl-b-D-glucoside and crystals of SARS-CoV-2 RBD-WCSL 129- PDI 96 grew in 0.1 M Tri-sodium citrate pH 5.5, 10% PEG8000. These crystals were flash frozen in mother liquor containing 20% glycerol or 20% 2-Methyl-2,4-pentanediol, respectively.

Hanging drop vapor diffusion crystallization trials were performed in-house for crystal optimization of SARS-CoV-2 RBD bound to WCSL 119, WCSL 129, WCSL 129- PDI 93, PDI 215, and PDI 231. Diffraction quality crystals of SARS-CoV-2 RBD-WCSL 119 crystals grew in 14% PEG3350, 0.2 M potassium thiocyanate seeded from initial screens and were stepwise transferred into cryo-protectant containing 30% ethylene glycol. SARS-CoV-2 RBD-WCSL 129 were obtained in 18% PEG3350, 0.1 M tri sodium citrate pH 5.5 seeded from initial screens and were harvested with 30% glycerol in mother liquor. A crystallization plate of SARS-CoV-2 RBD-PDI 215 was incubated at 4°C for 48 hours and subsequently transferred to 20°C. Crystals appeared after 2 days in 12% isopropanol, 12% PEG4000, 0.1 M tri sodium citrate pH 5.6 seeded from initial screens and were flash frozen in mother liquor containing 30% glycerol. SARS-CoV-2 RBD-WCSL 129- PDI 93 crystals grew in 23% PEG3350, 0.1 M sodium acetate pH 4.5 seeded from initial screens and SARS-CoV-2 RBD-PDI 231 crystals in 18% PEG3350, 10% N,N-Dimethyldodecylamine N-oxide (LDAO), 0.2 M sodium sulfate seeded from initial screens. Mother liquor containing 6% glycerol was used as cryo-protectant for SARS-CoV-2 RBD-WCSL 129-PDI 93 and mother liquor containing 20% butanediol was used for SARS-CoV-2 RBD-PDI 231 crystals.

X-ray diffraction data was collected at the MX2 beamline at the Australian Synchrotron, recorded with an Eiger 16M detector (Dectris) and processed using the XDS package (Kabsch, 2010). Molecular replacement using Phaser (Winn et al., 2011) was performed to solve the phase problem. SARS-CoV-2 RBD (PDB ID 6W41) and Fab structures of high sequence similarity were use as search models (PDB ID 6XC4 (heavy chain, HC) and 6UTA (light chain, LC) for PDI 37, for PDI 42, 7JXC for PDI 93, 3N9G for PDI 96, 5IES (heavy chain, HC) and 5WL2 (light chain, LC) for WCSL 119, 5HHV (HC) and 6A67 (LC) for WCSL 129, 6MHR for PDI 210, 6PE7 for PDI 215, 7CHB (HC) and 6PHB (LC) for PDI 231). For complexes of SARS-2 RBD bound by two Fabs simultaneously (WCSL 129-PDI 93, or WCSL 129-PDI 96) coordinates of the previously solved SARS-CoV-2 RBD-WCSL 129 structure was used as a search model instead of PDB ID 6W41. Iterative rounds of model building and refinement were undertaken using COOT (Emsley et al., 2010) and Phenix (Liebschner et al., 2019). Figures of the complexes were prepared using the PyMOL Molecular Graphics System, Version 2.3.0 (Schrödinger, LLC). Interactions, interfaces and buried areas from solvent were analyzed using PDBePISA v1.52 (Krissinel and Henrick, 2007). The atomic coordinates and structure factor files have been deposited in the Protein Data Bank. Accession numbers are listed in the crystallographic Table S3.