Crystals of N501 RBD/269 Fab were mounted in loops and dipped in solution containing 25% glycerol and 75% mother liquor for a second before being frozen in liquid nitrogen prior to data collection. No cryo-protectant was used for RBD/269 crystals. Diffraction data were collected at 100 K at beamline I03 of Diamond Light Source, UK. Diffraction images of 0.1° rotation were recorded on an Eiger2 XE 16M detector (exposure time of either 0.003 or 0.007 s per image, beam size 80 × 20 μm, 100% beam transmission and wavelength of 0.9763 Å). Data were indexed, integrated and scaled with the automated data processing program Xia2-dials (Winter, 2010; Winter et al., 2018). A dataset of 720° was collected from 2 frozen crystals to 2.19 Å resolution for N501Y RBD/269 Fab complex. 360° of data were collected for the RBD/269 Fab complex from a single crystal to 1.77 Å resolution.

Both structures were determined by molecular replacement with PHASER (McCoy et al., 2007) using search models of SARS-CoV-2 RBD/scFv269 complex (PDB: 7BEM) and the ChCl domains of SARS-CoV-2 RBD/158 complex (PDB: 7BEK) (Dejnirattisai et al., 2021). Cyclic model rebuilding with COOT (Emsley and Cowtan, 2004) and refinement with PHENIX (Liebschner et al., 2019) resulted in the current structures with Rwork/ Rfree = 0.197/ 0.222 and Rwork/ Rfree = 0.185/0.200 for all data to 2.19 Å and 1.77 Å resolution for N501Y RBD/269 Fab and RBD/269 Fab complexes, respectively. Electron density for the side chain of Y501 is weak. However, when the structure was refined with an asparagine at 501, there was strong, dispersed positive density around the side chain, consistent with the presence of a flexible tyrosine residue (Figure S2). Mass spectrometry and biolayer interferometry data confirmed the presence of tyrosine at 501. Data collection and structure refinement statistics are given in Table S1. Structural comparisons used SHP (Stuart et al., 1979), residues forming the RBD/Fab interface were identified with PISA (Krissinel and Henrick, 2007) and figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).

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