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- PDB-7tpr: Camel nanobodies 7A3 and 8A2 broadly neutralize SARS-CoV-2 variants -

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Basic information

Entry
Database: PDB / ID: 7tpr
TitleCamel nanobodies 7A3 and 8A2 broadly neutralize SARS-CoV-2 variants
Components
  • Nanobody 7A3
  • Nanobody 8A2
  • Spike glycoprotein
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / Neutralization / nanobody / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


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 ...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 / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / plasma membrane
Similarity search - Function
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 ...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
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Camelus dromedarius (Arabian camel)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsButay, K.J. / Zhu, J. / Dandey, V.P. / Hong, J. / Kwon, H.J. / Chen, C.Z. / Duan, Z. / Li, D. / Ren, H. / Liang, T. ...Butay, K.J. / Zhu, J. / Dandey, V.P. / Hong, J. / Kwon, H.J. / Chen, C.Z. / Duan, Z. / Li, D. / Ren, H. / Liang, T. / Martin, N. / Esposito, D. / Ortega-Rodriguez, U. / Xu, M. / Xie, H. / Ho, M. / Cachau, R. / Borgnia, M.J.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIA ES 103341 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 011943 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES 10326 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIC BC 011891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)Z01 BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)75N91019D00024 United States
CitationJournal: bioRxiv / Year: 2021
Title: Camel nanobodies broadly neutralize SARS-CoV-2 variants.
Authors: Jessica Hong / Hyung Joon Kwon / Raul Cachau / Catherine Z Chen / Kevin John Butay / Zhijian Duan / Dan Li / Hua Ren / Tianyuzhou Liang / Jianghai Zhu / Venkata P Dandey / Negin Martin / ...Authors: Jessica Hong / Hyung Joon Kwon / Raul Cachau / Catherine Z Chen / Kevin John Butay / Zhijian Duan / Dan Li / Hua Ren / Tianyuzhou Liang / Jianghai Zhu / Venkata P Dandey / Negin Martin / Dominic Esposito / Uriel Ortega-Rodriguez / Miao Xu / Mario J Borgnia / Hang Xie / Mitchell Ho /
Abstract: With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H nanobodies (7A3 and 8A2) from dromedary camels by phage display, ...With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants.
ONE-SENTENCE SUMMARY: Dromedary camel ( ) V H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.
History
DepositionJan 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
D: Nanobody 8A2
E: Nanobody 8A2
F: Nanobody 7A3
G: Nanobody 7A3
H: Nanobody 7A3


Theoretical massNumber of molelcules
Total (without water)449,7178
Polymers449,7178
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 126821.453 Da / Num. of mol.: 3
Mutation: F817P, A892P, A899P, A942P, K986P, V987P, and residues 682-685 from RRAR to GSAS
Source method: isolated from a genetically manipulated source
Details: Hexa-Pro construct mutations
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Antibody Nanobody 8A2


Mass: 13993.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli (E. coli) / Strain (production host): HB2151
#3: Antibody Nanobody 7A3


Mass: 13755.188 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli (E. coli) / Strain (production host): HB2151

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Ternary complex of SARS-Cov-2 Spike protein ectodomain (Wuhan strain) and camel nanobodies 7A3 and 8A2.COMPLEXall0MULTIPLE SOURCES
2SARS-CoV-2 Spike glycoproteinCOMPLEX#11RECOMBINANT
3Camel nanobody 8A2COMPLEX#21RECOMBINANT
4Camel nanobody 7A3COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.45 MDaNO
210.44 MDaNO
310.017 MDaNO
410.017 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Severe acute respiratory syndrome coronavirus 22697049
43Camelus dromedarius (Arabian camel)9838
54Camelus dromedarius (Arabian camel)9838
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCell
32Homo sapiens (human)9606Expi293F
43Escherichia coli (E. coli)562HB2151
54Escherichia coli (E. coli)562HB2151
Buffer solutionpH: 7.5 / Details: PBS
SpecimenConc.: 1.93 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Complexes of spike with 7A3 and/or 8A2 VHHs were prepared by mixing the components at a spike trimer to nanobody molar ratio of 1:6. The final concentration of spike trimer was 3 uM in PBS ...Details: Complexes of spike with 7A3 and/or 8A2 VHHs were prepared by mixing the components at a spike trimer to nanobody molar ratio of 1:6. The final concentration of spike trimer was 3 uM in PBS at pH 7 with the addition of 5 mM imidazole. Because the 8A2 stock was too diluted, complexes involving this nanobody were prepared at 0.5 uM spike trimer followed by a 6-fold concentration using a 10 kDa cutoff centrifugal filter (Amicon Ultra). All complexes were incubated on ice for at least 5 min prior to grid preparation.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company /
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Alignment procedure: ZEMLIN TABLEAU / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2200 nm / Nominal defocus min: 1500 nm / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDAccelerating voltage (kV)C2 aperture diameter (µm)Calibrated magnification (X)CryogenModelNominal magnification (X)
12006053648NITROGENFEI TALOS ARCTICA45000
23005047348FEI TITAN KRIOS81000
Image recording
IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Detector modeFilm or detector model
11160COUNTINGGATAN K2 SUMMIT (4k x 4k)
2260GATAN K3 BIOQUANTUM (6k x 4k)
Image scansSampling size: 5.000001 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryImaging-ID
2SerialEM3image acquisition1
4cryoSPARC3.3CTF correction
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
13cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1539054
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 580888 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00433397
ELECTRON MICROSCOPYf_angle_d0.57845472
ELECTRON MICROSCOPYf_dihedral_angle_d10.8044832
ELECTRON MICROSCOPYf_chiral_restr0.0455232
ELECTRON MICROSCOPYf_plane_restr0.0045822

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