Mcsg crystallization suite

The complex was concentrated to 10 mg/mL for initial crystal screening by sitting-drop vapor-diffusion in the MCSG Crystallization Suite (Anatrace, MCSG-1, MCSG-2, and MCSG-3) using a NT8 drop setter (Formulatrix). Diffracting crystals were obtained in a mother liquor (ML) containing 0.2 M (NH₄) Citrate, tribasic pH 7.0 and 12% (w/v) PEG 3350. The crystals were cryoprotected by soaking in ML supplemented with 30% (v/v) ethylene glycol. Diffraction data were collected at Advanced Photon Source SBC 19-ID at a 12.662 keV. The data set was processed using XDS^{22 (link)} and data reduction was performed using AIMLESS in CCP4 to a resolution of 2.75 Å. The structure of the complex was solved by molecular replacement using Phaser^{23 (link)} in Phenix^{24 (link)} with a search model of SARS-CoV-2 RBD (PDBid: 6LZG)^{9 (link)} and the Fab structure (PDBid: 5I1E)^{25 (link)} divided into Fv and Fc portions. Remaining model building was completed using COOT^{26 (link)} and refinement was performed in Phenix^{24 (link)}. The data collection and refinement statistics are summarized in Supplementary Table 1. Structural figures were made in PyMol (Schrodinger, LLC).

CV3-25 Fab was incubated with a 1.5 molar excess of the synthetic stem helix peptide spanning residues 1149–1167 (Genscript). Initial crystal screening was performed by sitting-drop vapor-diffusion in the MCSG Crystallization Suite (Anatrace) using a NT8 drop setter (Formulatrix). Poorly diffracting crystals grew in MCSG-3 well B1 and were optimized using the Additive Screen (Hampton Scientific). Diffracting crystals were obtained in a mother liquor (ML) containing 0.1 M Na Acetate:HCl, pH 4.5, 2.0 M (NH₄)SO₄, 0.1 M Strontium Chloride. The crystals were cryoprotected by soaking in ML supplemented with 26% glycerol. Diffraction data were collected at Advanced Light Source beamline 5.0.2 at 12286 keV. The data set was processed using XDS^{88 (link)} and data reduction was performed using AIMLESS in CCP4^{89 (link)} to a resolution of 1.74 Å. Initial phases were solved by molecular replacement using Phaser^{90 (link)} in Phenix^{91 (link),92 (link)} with a search model of Fab 4AB007 (PDBid: 5MVZ) divided into Fv and Fc portions. Model building was completed using COOT^{93 (link)} and refinement was performed in Phenix with the final refinement run through the PDB_REDO server^{94 (link)}. The data collection and refinement statistics are summarized in Table 1. Structural figures were made in Pymol (Schrodinger, LLC).