Nt8 drop setter

The complex was concentrated to 10mg/mL for initial crystal screening by sitting-drop vapor-diffusion in the MCSG Suite (Anatrace) using a NT8 drop setter (Formulatrix). Diffracting crystals were obtained in a mother liquor (ML) containing 0.2M (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 was collected at Advanced Photon Source (APS) SBC 19-ID at a 12.662 keV. The data set was processed using XDS^{17 (link)} to a resolution of 2.75Å. The structure of the complex was solved by molecular replacement using Phaser^{18 (link)} with a search model of SARS-CoV-2 RBD (PDBid: 6lzg)⁹ and the Fab structure (PDBid: 5i1e)^{19 (link)} divided into Fv and Fc portions. Remaining model building was completed using COOT^{20 (link)} and refinement was performed in Phenix^{21 (link)}. The data collection and refinement statistics are summarized in Extended Data Table 1. Structural figures were made in Pymol.

G3BP1 NTF2 TripleW crystals were obtained by vapor diffusion in a sitting drop composed of 200 nl of 1.6 mg/ml protein in 100 mM NaCl, 20 mM Tris, pH 8.5, and 200 nl of well solution (20% [w/v] PEG 8000, 100 mM HEPES pH 7.5) and equilibrated by vapor diffusion with 50 μl of well solution. The drop was set up using a NT8 drop setter (Formulatrix) and the well solution was from the Wizard Classic I&II High-Throughput Screen (Molecular Dimensions). The tray was incubated and imaged using Rock Imager and Rock Maker instrumentation (Formulatrix). The crystal was harvested straight from the screening drop without cryoprotectant or oil and immediately flash cooled in liquid nitrogen. The rotation data collection was performed at the National Synchrotron Light Source II beamline 17-ID-2 (FMX) equipped with an Eiger 16M detector at 100 K, a rotation range of 0.2° per frame, and a total of wedge of 180°. The diffraction images were processed using XDS (79 (link)). The R_free set was generated from 5% of the reflections in thin resolution shells using the Phenix (80 (link)) reflection file editor. Initial phases were generated by Phaser (81 (link)) via molecular replacement using G3BP1 NTF2 WT (PDB ID 4FCJ) as a search model. Iterative automatic and manual refinement was performed using Phenix and Coot (80 (link), 82 (link)).

Initial crystallization screening was performed by sitting-drop vapor-diffusion using the MCSG Crystallization Suite (Anatrace) using an NT8 drop setter (Formulatrix). Poorly formed crystals grew in MCSG3 well D6 (0.1 M MES, pH 6.0, 0.2 M Zn acetate, 10% PEG 8K). Crystals were optimized using the Additive Screen HT (Hampton Research). Well diffracting crystals were grown via hanging-drop vapor-diffusion using the same condition with the addition of 5% 1-propanol and were frozen with 30% glycerol as a cryoprotectant. Diffraction data were collected at Advanced Light Source beamline 5.0.2 at 12.731keV. The dataset was processed using XDS [55 (link)] and data reduction was performed using AIMLESS in CCP4 [56 (link)] to a resolution of 2.68 Å. Initial phases were solved by molecular replacement using Phaser in Phenix [57 (link)] with a search model of HB3VAR03 (PDB ID 4V3D) and mAb 258259 (PDB ID 6WTV) divided into Fv and CH1 domains. Model building was completed using Coot [58 (link)] and refinement was performed in Phenix. Additional model refinement was done using ISOLDE [59 (link)] in ChimeraX [60 (link)]. The final refinement was performed using PDB-REDO server [61 ]. Data collection and refinement statistics are summarized in Table S3.