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- EMDB-11218: H11-H4 bound to Spike -

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Basic information

Entry
Database: EMDB / ID: EMD-11218
TitleH11-H4 bound to Spike
Map data
SampleComplex between Spike and nanobody H11-H4:
Spike / nanobody H11-H4 / Spike glycoprotein,Fibritin / Nanobody H11-H4 / ligand
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / host cell surface receptor binding / virion / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane
Fibritin C-terminal / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike receptor binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus
Spike glycoprotein / Fibritin
Biological speciesEnterobacteria phage T4 (bacteriophage) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsClare DK / Naismith JH / Weckener M / Vogirala VK
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust100209/Z/12/Z United Kingdom
Engineering and Physical Sciences Research CouncilRosalind Franklin United Kingdom
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2020
Title: Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.
Authors: Jiangdong 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: Jiangdong 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 ReportPDB-ID: 6zhd

SummaryFull reportAbout validation report
History
DepositionJun 22, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 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: 0.009
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6zhd
  • 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_11218.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 318. Å
1.06 Å/pix.
x 300 pix.
= 318. Å
1.06 Å/pix.
x 300 pix.
= 318. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.009 / Movie #1: 0.009
Minimum - Maximum-0.0153223965 - 0.047560856
Average (Standard dev.)0.0000410761 (±0.0019229134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z318.000318.000318.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0150.0480.000

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Supplemental data

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Sample components

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Entire Complex between Spike and nanobody H11-H4

EntireName: Complex between Spike and nanobody H11-H4 / Details: Nanobody complexed to Spike protein / Number of components: 6

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Component #1: protein, Complex between Spike and nanobody H11-H4

ProteinName: Complex between Spike and nanobody H11-H4 / Details: Nanobody complexed to Spike protein / Recombinant expression: No
MassTheoretical: 520 kDa

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Component #2: protein, Spike

ProteinName: Spike / Recombinant expression: No
SourceSpecies: Enterobacteria phage T4 (bacteriophage)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, nanobody H11-H4

ProteinName: nanobody H11-H4 / Recombinant expression: No
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Spike glycoprotein,Fibritin

ProteinName: Spike glycoprotein,Fibritin / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 142.399375 kDa
SourceSpecies: Enterobacteria phage T4 (bacteriophage)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Nanobody H11-H4

ProteinName: Nanobody H11-H4 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 15.045752 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.: 1 mg/mL / Buffer solution: 50 mM Tris, pH 7, 150 mM NaCl / pH: 7
Support filmPlasma Cleaner PDC-002-CE, Harrick Plasma
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 81 %
Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 90 s (Plasma Cleaner PDC- ...Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 90 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) to activate the nanowires. Approximately 6 nL of the complex were applied to the grids using a Chameleon EP system (SPT Labtech) at 81 % relative humidity and ambient temperature..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: TFS KRIOS / Details: EPU auto-function coma free correction
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 46 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000 X (nominal), 47170 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 3000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (100.0 - 100.0 K)
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 10000 / Sampling size: 5 µm / Details: The images were collected as 50 frame movies

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 126938 / Details: Images were aligned and reconstructed in Releion
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / CTF correction: Relion / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Relion / Euler angles: Relion
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Correlation / Refinement space: RECIPROCAL / Details: REFMAC was used via ccpem GUI
Input PDB model: 6Z43
Output model

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