EMDB-14591: CryoEM structure of SARS-CoV-2 spike monomer in complex with neut...

Basic information

Entry

Database: EMDB / ID: EMD-14591

• Title: CryoEM structure of SARS-CoV-2 spike monomer in complex with neutralising antibody P008_60
• Map data: DeepEMenhancer map

Sample

• Complex: Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in complex with a neutralising antibody P008_60
  • Protein or peptide: Spike glycoproteinSpike protein
  • Protein or peptide: P008_60 antibody, Heavy chain
  • Protein or peptide: P008_60 antibody, Light chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
• Ligand: 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

Function / homology

Function:

Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane

Domain/homology:

Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal

Component:

Spike glycoprotein

• Biological species: Homo sapiens (human) / Severe acute respiratory syndrome coronavirus 2
• Method: single particle reconstruction / cryo EM / Resolution: 4.31 Å
• Authors: Rosa A / Pye VE / Cronin N / Cherepanov P

Funding support

United Kingdom, 5 items

Organization	Grant number	Country
The Francis Crick Institute	FC001061	United Kingdom
Wellcome Trust	FC001061	United Kingdom
Medical Research Council (MRC, United Kingdom)	FC001061	United Kingdom
Cancer Research UK	FC001061	United Kingdom
Wellcome Trust	206175/Z/17/Z	United Kingdom

• Citation: 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 / 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 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.

History

Deposition	Mar 24, 2022	-
Header (metadata) release	Aug 17, 2022	-
Map release	Aug 17, 2022	-
Update	Aug 31, 2022	-
Current status	Aug 31, 2022	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_14591.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: DeepEMenhancer map
• Voxel size: X=Y=Z: 1.1 Å

Density

Contour Level	By AUTHOR: 0.03
Minimum - Maximum	-0.0017089208 - 1.8422732
Average (Standard dev.)	0.0004936652 (±0.015751675)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 340 340 340 Spacing 340 340 340
Cell	A=B=C: 374.0 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_14591_msk_1.map

Additional map: original map, no modifications

• File: emd_14591_additional_1.map
• Annotation: original map, no modifications

Half map: half map A

• File: emd_14591_half_map_1.map
• Annotation: half map A

Half map: half map B

• File: emd_14591_half_map_2.map
• Annotation: half map B

Sample components

Entire : Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in comple...

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

Components

• Complex: Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in complex with a neutralising antibody P008_60
  • Protein or peptide: Spike glycoproteinSpike protein
  • Protein or peptide: P008_60 antibody, Heavy chain
  • Protein or peptide: P008_60 antibody, Light chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
• Ligand: 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

Supramolecule #1: Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in comple...

• Supramolecule: Name: Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in complex with a neutralising antibody P008_60; type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 125 KDa

Macromolecule #1: Spike glycoprotein

• Macromolecule: Name: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus 2
• Molecular weight: Theoretical: 142.269859 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MGILPSPGMP ALLSLVSLLS VLLMGCVAET GMFVFLVLLP LVSSQCVNLT TRTQLPPAYT NSFTRGVYYP DKVFRSSVLH STQDLFLPF FSNVTWFHAI HVSGTNGTKR FDNPVLPFND GVYFASTEKS NIIRGWIFGT TLDSKTQSLL IVNNATNVVI K VCEFQFCN DPFLGVYYHK NNKSWMESEF RVYSSANNCT FEYVSQPFLM DLEGKQGNFK NLREFVFKNI DGYFKIYSKH TP INLVRDL PQGFSALEPL VDLPIGINIT RFQTLLALHR SYLTPGDSSS GWTAGAAAYY VGYLQPRTFL LKYNENGTIT DAV DCALDP LSETKCTLKS FTVEKGIYQT SNFRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLY NSASF STFKCYGVSP TKLNDLCFTN VYADSFVIRG DEVRQIAPGQ TGKIADYNYK LPDDFTGCVI AWNSNNLDSK VGGNY NYLY RLFRKSNLKP FERDISTEIY QAGSTPCNGV EGFNCYFPLQ SYGFQPTNGV GYQPYRVVVL SFELLHAPAT VCGPKK STN LVKNKCVNFN FNGLTGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGT NTSNQVA VL YQDVNCTEVP VAIHADQLTP TWRVYSTGSN VFQTRAGCLI GAEHVNNSYE CDIPIGAGIC ASYQTQTNSP SRASSVAS Q SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTG IAVEQDKNTQ EVFAQVKQIY KTPPIKDFGG FNFSQILPDP SKPSKRSFIE DLLFNKVTLA DAGFIKQYGD CLGDIAARDL ICAQKFNGL TVLPPLLTDE MIAQYTSALL AGTITSGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE NQKLIANQFN S AIGKIQDS LSSTASALGK LQDVVNQNAQ ALNTLVKQLS SNFGAISSVL NDILSRLDPP EAEVQIDRLI TGRLQSLQTY VT QQLIRAA EIRASANLAA TKMSECVLGQ SKRVDFCGKG YHLMSFPQSA PHGVVFLHVT YVPAQEKNFT TAPAICHDGK AHF PREGVF VSNGTHWFVT QRNFYEPQII TTDNTFVSGN CDVVIGIVNN TVYDPLQPEL DSFKEELDKY FKNHTSPDVD LGDI SGINA SVVNIQKEID RLNEVAKNLN ESLIDLQELG KYEQSGRENL YFQGGGGSGY IPEAPRDGQA YVRKDGEWVL LSTFL GHHH HHH

Macromolecule #2: P008_60 antibody, Heavy chain

• Macromolecule: Name: P008_60 antibody, Heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.954072 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

EVQLVESGGG VVQPGRSLRL TCAASGFIFS SYGMHWVRQA PGKGLEWVAV ISYDGSYKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCTKA DYYDFWSGYQ KTYYYYMDVW GKGTTVTISS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK D YFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVG RTKHHHHHH

Macromolecule #3: P008_60 antibody, Light chain

• Macromolecule: Name: P008_60 antibody, Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 23.589068 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

DIQLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GTSSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYC QQYGSSPQIT FGQGTRLEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 8 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

Macromolecule #5: 3-[5-[(4-ethyl-3-methyl-5-oxidanylidene-pyrrol-2-yl)methyl]-2-[[5...

• Macromolecule: Name: 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; type: ligand / ID: 5 / Number of copies: 1 / Formula: 3Q9
• Molecular weight: Theoretical: 588.694 Da

Chemical component information

ChemComp-3Q9:
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

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.5 mg/mL
• Buffer: pH: 8 ; Details: 0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH8.0
• Grid: Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: 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

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.7000000000000001 µm
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• 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/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• 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, 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
• Initial 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 populated class was selected, resulting in the final set of 166,619 particles.
• Final angle assignment: Type: PROJECTION MATCHING
• 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
• Details: 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

Atomic model buiding 1

Initial model

PDB ID	Chain
7a92	chain_id: A
5wi9	chain_id: E
5wi9	chain_id: F

• Details: 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