CryoEM structure of SARS-CoV-2 spike monomer in complex with neutralising antibody P008_60

3-[5-[(4-ethyl-3-methyl-5-oxidanylidene-pyrrol-2-yl)methyl]-2-[[5-[(3-ethyl-4-methyl-5-oxidanylidene-pyrrol-2-yl)methyl]-3-(3-hydroxy-3-oxopropyl)-4-methyl-1H-pyrrol-2-yl]methyl]-4-methyl-1H-pyrrol-3-yl]propanoic acid

N-acetyl-beta-D-glucosamine; 2-acetamido-2-deoxy-beta-D-glucose; 2-acetamido-2-deoxy-D-glucose; 2-acetamido-2-deoxy-glucose; N-ACETYL-D-GLUCOSAMINE

A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.

The atomistic models of monomeric SARS-CoV-2 S1 protein and a Fab molecule, extracted from PDB entries 7A92 and 5WI9, were docked in the cryo-EM map using Chimera. The NTD and the RBD of the spike subunit were replaced with the crystal structures from PDB entries 7B62 and 7OAO, respectively. Guided by the cryo-EM map, the model was adjusted and extended interactively in Coot and refined using phenix.real_space_refine.

0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH8.0

Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in complex with a neutralising antibody P008_60

The micrograph stacks were aligned, binned to the physical pixel size of 1.1 A and summed, with dose weighting applied, as implemented in MotionCor2

Initially, particles were picked with Gautomatch-v0.53 using 2D class averages of the trimeric spike, low-pass filtered to 20 A resolution, as templates. The resulting 1,740,430 particles, extracted in Relion-3.1 and binned to a pixel size of 4.4 A, were subjected to two rounds of reference-free 2D classification in cryoSPARC-2. 283,956 particles belonging to well-defined 2D classes were subjected to classification into twelve 3D classes in Relion-3.1. Neither the 2D nor 3D class averages of the trimeric spike revealed features attributable to a bound Fab molecule. Next, 3,772,722 particles were picked using 2D class averages of the dissociated spikes. Following two rounds of 2D classification in cryoSPARC-2, 753,837 particles, re-extracted with pixel size of 2.2 A, were subjected to 3D classification in Relion-3.1 into 9 classes using an initial model obtained by Ab-initio reconstruction in cryoSPARC-2. The procedure revealed a single well-defined 3D class containing 208,343 particles (27.4%) of S1 protein with a bound Fab molecule. The particles, re-extracted without binning (with a pixel size 1.1 A), were subjected to two rounds of 3D classification using Ab-initio reconstruction in cryoSPARC-2 with 2 classes and class similarity set to 0. At the end of each round, the most populated class was selected, resulting in the final set of 166,619 particles.

SARS-CoV-2 spike ectodomain (0.5 mg/ml), supplemented 0.2 mg/ml P008_60 Fab and 0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH8.0

Severe acute respiratory syndrome coronavirus 2

Medical Research Council (MRC, United Kingdom)

covid-19, SARS-CoV-2, spike, neutralizing antibody, immunity, VIRAL PROTEIN