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- PDB-8w4f: SARS-CoV-2 spike protein in complex with a trivalent nanobody -

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Basic information

Entry
Database: PDB / ID: 8w4f
TitleSARS-CoV-2 spike protein in complex with a trivalent nanobody
Components
  • Spike glycoprotein
  • Tribody
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / trivalent nanobody / viral neutralization / all-RBD-down spike / 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 / entry receptor-mediated virion attachment to host cell ...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 / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / membrane fusion / Attachment and Entry / 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
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
Model detailsMODEL GENERATED BY ROSETTA VERSION 2022.11+release.512e589
AuthorsJiang, X.Y. / Qian, J.Q. / Zhu, H.X. / Qin, Q. / Huang, Q.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971377 China
National Natural Science Foundation of China (NSFC)31671386 China
CitationJournal: Int J Biol Macromol / Year: 2024
Title: Structure-guided design of a trivalent nanobody cluster targeting SARS-CoV-2 spike protein.
Authors: Xinyi Jiang / Qin Qin / Haixia Zhu / Jiaqiang Qian / Qiang Huang /
Abstract: Nanobodies are natural anti-SARS-CoV-2 drug candidates. Engineering multivalent nanobodies is an effective way to improve the functional binding affinity of natural nanobodies by simultaneously ...Nanobodies are natural anti-SARS-CoV-2 drug candidates. Engineering multivalent nanobodies is an effective way to improve the functional binding affinity of natural nanobodies by simultaneously targeting multiple sites on viral proteins. However, multivalent nanobodies have usually been engineered by trial and error, and rational designs are still lacking. Here, we describe a structure-guided design of a self-assembled trivalent nanobody cluster targeting the SARS-CoV-2 spike protein. Using the nanobody Nb6 as a monovalent binder, we first selected a human-derived trimerization scaffold evaluated by molecular dynamics simulations, then selected an optimal linker according to the minimum distance between Nb6 and the trimerization scaffold, and finally successfully engineered a trivalent nanobody cluster called Tribody. Compared with the low-affinity monovalent counterpart (Nb6), Tribody showed much higher target binding affinity (K < 1 pM) and thus had a 900-fold increase in antiviral neutralization against SARS-CoV-2 pseudovirus. We determined the cryo-EM structure of the Tribody-spike complex and confirmed that all three Nb6 binders of Tribody collectively bind to the three receptor-binding domains (RBDs) of the spike and lock them in a 3-RBD-down conformation, fully consistent with our structure-guided design. This study demonstrates that synthetic nanobody clusters with human-derived self-assembled scaffolds are potential protein drugs against SARS-CoV-2 coronaviruses.
History
DepositionAug 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 24, 2024Provider: 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: Tribody
E: Tribody
F: Tribody


Theoretical massNumber of molelcules
Total (without water)434,3156
Polymers434,3156
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 123893.242 Da / Num. of mol.: 3 / Mutation: R683A/R685A/F817P/A892P/A899P/A942P/R986P/V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Antibody Tribody


Mass: 20878.377 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: a trivalent Nb6 construct / Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SARS-CoV-2 spike protein in complex with a trivalent nanobodyCOMPLEXall0RECOMBINANT
2SARS-CoV-2 spike proteinCOMPLEX#11RECOMBINANT
3trivalent nanobodyCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Severe acute respiratory syndrome coronavirus 22697049
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562Rosetta(DE3)
Buffer solutionpH: 8 / Details: 20 mM Tris,150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl) aminomethaneC4H11NO31
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 56 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.1.2particle selection
4CTFFIND4CTF correction
10cryoSPARC4.1.2initial Euler assignment
11cryoSPARC4.1.2final Euler assignment
12cryoSPARC4.1.2classification
13cryoSPARC4.1.23D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2754775
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 262855 / Symmetry type: POINT

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