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- EMDB-11068: Cryo-EM Structure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11068
TitleCryo-EM Structure of SARS-CoV-2 Spike : H11-D4 Nanobody Complex
Map data
SampleSpike glycoprotein in complex with H11-D4 nanobodiesPeplomer:
(Spike glycoproteinPeplomer) x 2 / (NanobodySingle-domain antibody) 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 / pathogenesis / 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 / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRuza RR / Duyvesteyn HME / Shah P / Carrique L / Ren J / Malinauskas T / Zhou D / Stuart DI / Naismith JH
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilEP/S025243/1 United Kingdom
Cancer Research UKC20724/A14414 United Kingdom
Wellcome Trust100209/Z/12/Z United Kingdom
Wellcome Trust101122/Z/13/Z United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.
Authors: Jiandong Huo / Audrey Le Bas / Reinis R Ruza / Helen M E Duyvesteyn / Halina Mikolajek / Tomas Malinauskas / Tiong Kit Tan / Pramila Rijal / Maud Dumoux / Philip N Ward / Jingshan Ren / ...Authors: Jiandong Huo / Audrey Le Bas / Reinis R Ruza / Helen M E Duyvesteyn / Halina Mikolajek / Tomas Malinauskas / Tiong Kit Tan / Pramila Rijal / Maud Dumoux / Philip N Ward / Jingshan Ren / Daming Zhou / Peter J Harrison / Miriam Weckener / Daniel K Clare / Vinod K Vogirala / Julika Radecke / Lucile Moynié / Yuguang Zhao / Javier Gilbert-Jaramillo / Michael L Knight / Julia A Tree / Karen R Buttigieg / Naomi Coombes / Michael J Elmore / Miles W Carroll / Loic Carrique / Pranav N M Shah / William James / Alain R Townsend / David I Stuart / Raymond J Owens / James H Naismith /
Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the ...The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 22, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z43
  • Surface level: 0.01
  • 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_11068.map.gz / Format: CCP4 / Size: 561.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 528 pix.
= 438.24 Å
0.83 Å/pix.
x 528 pix.
= 438.24 Å
0.83 Å/pix.
x 528 pix.
= 438.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.10291079 - 0.17046817
Average (Standard dev.)0.0000813213 (±0.002073685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions528528528
Spacing528528528
CellA=B=C: 438.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z528528528
origin x/y/z0.0000.0000.000
length x/y/z438.240438.240438.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS528528528
D min/max/mean-0.1030.1700.000

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Supplemental data

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Sample components

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Entire Spike glycoprotein in complex with H11-D4 nanobodies

EntireName: Spike glycoprotein in complex with H11-D4 nanobodiesPeplomer
Number of components: 6

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Component #1: protein, Spike glycoprotein in complex with H11-D4 nanobodies

ProteinName: Spike glycoprotein in complex with H11-D4 nanobodiesPeplomer
Recombinant expression: No
MassTheoretical: 480 kDa

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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) / Vector: pHR-CMV-TetO2 / Cell of expression system: HEK293S GnTI-

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Component #3: protein, Nanobody

ProteinName: NanobodySingle-domain antibody / Recombinant expression: No
SourceSpecies: Lama glama (llama)
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, Nanobody

ProteinName: NanobodySingle-domain antibody / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 16.679453 kDa
SourceSpecies: Lama glama (llama)
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: 26 / 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 solutionSpecimen conc.: 0.25 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.65 K / Humidity: 100 % / Details: Blot for 6 seconds with a blotting force of -1.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 42.8 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
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: 305513
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 6VXX
Output model

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