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- EMDB-11616: Cryo-EM structure of the SARS-CoV-2 spike protein bound to neutra... -

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Basic information

Entry
Database: EMDB / ID: EMD-11616
TitleCryo-EM structure of the SARS-CoV-2 spike protein bound to neutralizing sybodies (Sb23)
Map data
SampleSARS-CoV-2 spike glycoprotein in 1-up conformation bound by 3 neutralising Sybodies (Sb23):
(Spike glycoproteinPeplomer) x 2 / Neutralising sybody (Sb23) / Sybody 23 / ligand
Function / homology
Function and homology information


suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / viral translation / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / viral protein processing ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / viral translation / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / viral protein processing / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / go:0009405: / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Spike receptor binding domain superfamily, coronavirus / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / in:ipr027400: / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like
Spike glycoprotein
Biological speciesSevere acute respiratory syndrome coronavirus 2 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsHallberg BM / Das H
Citation
Journal: Nat Commun / Year: 2020
Title: Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.
Authors: Tânia F Custódio / Hrishikesh Das / Daniel J Sheward / Leo Hanke / Samuel Pazicky / Joanna Pieprzyk / Michèle Sorgenfrei / Martin A Schroer / Andrey Yu Gruzinov / Cy M Jeffries / Melissa ...Authors: Tânia F Custódio / Hrishikesh Das / Daniel J Sheward / Leo Hanke / Samuel Pazicky / Joanna Pieprzyk / Michèle Sorgenfrei / Martin A Schroer / Andrey Yu Gruzinov / Cy M Jeffries / Melissa A Graewert / Dmitri I Svergun / Nikolay Dobrev / Kim Remans / Markus A Seeger / Gerald M McInerney / Ben Murrell / B Martin Hällberg / Christian Löw /
Abstract: The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional ...The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
#1: Journal: BiorXiv / Year: 2020
Title: Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2
Authors: Custodio TF / Das H / Sheward DJ / Hanke L / Pazicky S / Pieprzyk J / Sorgenfrei M / Schroer M / Gruzinov A / Jeffries C / Graewert M / Svergun D / Dobrev N / Remans K / Seeger MA / ...Authors: Custodio TF / Das H / Sheward DJ / Hanke L / Pazicky S / Pieprzyk J / Sorgenfrei M / Schroer M / Gruzinov A / Jeffries C / Graewert M / Svergun D / Dobrev N / Remans K / Seeger MA / McInerney GM / Murrell B / Hallberg BM / Loew C
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 16, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a25
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11616.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 600 pix.
= 612. Å
1.02 Å/pix.
x 600 pix.
= 612. Å
1.02 Å/pix.
x 600 pix.
= 612. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.83219665 - 1.8782024
Average (Standard dev.)-0.0002558347 (±0.022415627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 612.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z612.000612.000612.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.8321.878-0.000

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Supplemental data

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Additional map: Map used for refinement. Generated through the use...

Fileemd_11616_additional_1.map
AnnotationMap used for refinement. Generated through the use of ResolveCryoEM in Phenix using the half maps.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Local reconstruction of the interface between chain C...

Fileemd_11616_additional_2.map
AnnotationLocal reconstruction of the interface between chain C and F. The density is translated vs the main map. Improved density for the Sb23 RBD interface compared to the main map. To be used in modelling.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_11616_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_11616_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire SARS-CoV-2 spike glycoprotein in 1-up conformation bound by 3 neu...

EntireName: SARS-CoV-2 spike glycoprotein in 1-up conformation bound by 3 neutralising Sybodies (Sb23)
Number of components: 6

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Component #1: protein, SARS-CoV-2 spike glycoprotein in 1-up conformation bound...

ProteinName: SARS-CoV-2 spike glycoprotein in 1-up conformation bound by 3 neutralising Sybodies (Sb23)
Recombinant expression: No

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Component #2: protein, Spike glycoprotein

ProteinName: Spike glycoproteinPeplomer / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, Neutralising sybody (Sb23)

ProteinName: Neutralising sybody (Sb23) / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Spike glycoprotein

ProteinName: Spike glycoproteinPeplomer / Details: Same as 6VSB / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 126.697531 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Sybody 23

ProteinName: Sybody 23 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 12.532897 kDa
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: ligand, 2-acetamido-2-deoxy-beta-D-glucopyranose

LigandName: 2-acetamido-2-deoxy-beta-D-glucopyranose / Number of Copies: 45 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 300.0 - 1100.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 70854
3D reconstructionSoftware: cryoSPARC / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Output model

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