Journal: Cell Rep / Year: 2022 Title: A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination. Authors: Jeffrey Seow / Hataf Khan / Annachiara Rosa / Valeria Calvaresi / Carl Graham / Suzanne Pickering / Valerie E Pye / Nora B Cronin / Isabella Huettner / Michael H Malim / Argyris Politis / ...Authors: Jeffrey Seow / Hataf Khan / Annachiara Rosa / Valeria Calvaresi / Carl Graham / Suzanne Pickering / Valerie E Pye / Nora B Cronin / Isabella Huettner / Michael H Malim / Argyris Politis / Peter Cherepanov / Katie J Doores / Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3 to 4 sec before plunging.
Details
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
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 16624 / Average exposure time: 4.1 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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
Particle selection
Number selected: 1740430 Details: 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, ...Details: 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.
CTF correction
Software - Name: Gctf (ver. 1.06)
Startup model
Type of model: NONE Details: Starting model was generating using ab-initio procedure in cryoSPARC
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Details: Non Uniform refinement in cryoSPARC / Number images used: 166619
Initial angle assignment
Type: PROJECTION MATCHING
Final angle assignment
Type: PROJECTION MATCHING
Final 3D classification
Number classes: 2 / Software - Name: RELION (ver. 3.1) Details: Particles 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 ...Details: Particles 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.
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.
Refinement
Space: REAL / Protocol: OTHER / Overall B value: 81 / Target criteria: Correlation coefficient
Output model
PDB-7zbu: CryoEM structure of SARS-CoV-2 spike monomer in complex with neutralising antibody P008_60
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