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- EMDB-11617: Cryo-EM structure of the SARS-CoV-2 spike protein bound to neutra... -

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Basic information

Entry
Database: EMDB / ID: EMD-11617
TitleCryo-EM structure of the SARS-CoV-2 spike protein bound to neutralizing sybodies (Sb23) 2-up conformation
Map data
SampleSARS-CoV-2 spike glycoprotein in 1-up conformation bound by 3 neutralising Sybodies (Sb23):
(Spike glycoproteinPeplomer) x 2 / (Neutralising sybody ...) x 2 / 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 / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / go:0009405: / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike glycoprotein S2, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S1, C-terminal
Spike glycoprotein
Biological speciesSevere acute respiratory syndrome coronavirus 2 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsHallberg BM / Das H
CitationJournal: 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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 16, 2020-
Header (metadata) releaseOct 21, 2020-
Map releaseOct 21, 2020-
UpdateNov 18, 2020-
Current statusNov 18, 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-7a29
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11617.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: 0.174 / Movie #1: 0.2
Minimum - Maximum-0.9396764 - 2.3658695
Average (Standard dev.)-0.00031092158 (±0.025100965)
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/NZ9482110
NX/NY/NZ11313776
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.9402.366-0.000

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Supplemental data

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Additional map: Locally refined focussed map on the C and...

Fileemd_11617_additional_1.map
AnnotationLocally refined focussed map on the C and F chain interaction. ResolveCryoEM from the Phenix suite generated the map from the respective half maps in the local refinement in CryoSparc.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_11617_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_11617_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 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 142.399375 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Neutralising sybody (Sb23)

ProteinName: Neutralising sybody (Sb23) / 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 / Number of projections: 69567
3D reconstructionSoftware: cryoSPARC / Resolution: 2.94 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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