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- PDB-7r4q: The SARS-CoV-2 spike in complex with the 1.29 neutralizing nanobody -

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Basic information

Entry
Database: PDB / ID: 7r4q
TitleThe SARS-CoV-2 spike in complex with the 1.29 neutralizing nanobody
Components
  • Camel-derived nanobody 1.29
  • Spike glycoproteinSpike protein
KeywordsVIRAL PROTEIN / coronavirus / COVID-19 / SARS-CoV-2 / spike / nanobodies / neutralization
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 / membrane / identical protein binding / 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: 3.6 Å
AuthorsCasasnovas, J.M. / Melero, R. / Arranz, R. / Fernandez, L.A.
Funding support Spain, European Union, 2items
OrganizationGrant numberCountry
Ministry of Economy and Competitiveness (MINECO)202020E079 Spain
European Union (EU)ESFRI-2019-01-CSIC-16European Union
CitationJournal: Front Immunol / Year: 2022
Title: Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron.
Authors: José M Casasnovas / Yago Margolles / María A Noriega / María Guzmán / Rocío Arranz / Roberto Melero / Mercedes Casanova / Juan Alberto Corbera / Nereida Jiménez-de-Oya / Pablo ...Authors: José M Casasnovas / Yago Margolles / María A Noriega / María Guzmán / Rocío Arranz / Roberto Melero / Mercedes Casanova / Juan Alberto Corbera / Nereida Jiménez-de-Oya / Pablo Gastaminza / Urtzi Garaigorta / Juan Carlos Saiz / Miguel Ángel Martín-Acebes / Luis Ángel Fernández /
Abstract: The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish ...The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.
History
DepositionFeb 9, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 8, 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: Camel-derived nanobody 1.29
E: Camel-derived nanobody 1.29
hetero molecules


Theoretical massNumber of molelcules
Total (without water)456,46143
Polymers447,0395
Non-polymers9,42238
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 140113.922 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell (production host): HEK293F
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
References: UniProt: P0DTC2
#2: Antibody Camel-derived nanobody 1.29


Mass: 13348.559 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Plasmid: pcDNA
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 33 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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
IDNameTypeDetailsEntity IDParent-IDSource
1The trimeric SARS-CooV-2 spike in complex with the 1.29 neutralizing nanobodyCOMPLEXComplex of a soluble spike of the SARS-CoV-2 stabilized in the prefusion form with the 1.29 nanobody bound to its RBD#1-#20RECOMBINANT
2SARS-CoV-2 spikeCOMPLEXTrimeric spike in its prefusion conformation#11RECOMBINANT
3Nanobody 1.29COMPLEXImmunoglobulin domain#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.2 MDaYES
210.19 MDaYES
3110.0 kDa/nmYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Severe acute respiratory syndrome coronavirus 22697049
32Severe acute respiratory syndrome coronavirus 22697049
43Camelus dromedarius (Arabian camel)9838
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
21Mammalian expression vector Flag-MCS-pcDNA3.1 (others)2021188HEK293F
32Mammalian expression vector Flag-MCS-pcDNA3.1 (others)2021188HEK293F
43Mammalian expression vector Flag-MCS-pcDNA3.1 (others)2021188HEK293F
Buffer solutionpH: 7.7
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM2-amino-2-(hydroxymethyl)propane-1,3-diolTris-HClTris1
2200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The spike with the bound nanobody was purified by size exclusion chromatography.
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_4335: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.15model fitting
8Coot0.8.9.1model fitting
14PHENIX1.17.1_3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40000 / Symmetry type: POINT
Atomic model buildingB value: 140 / Protocol: OTHER / Space: REAL / Target criteria: correlation coefficient
Details: Rigid body refinement. Real space refinement including 5 macro cycles of:Minimization_global, local_grid search and adp. Refinement at 4.0A of resolution. Refinement included NCS.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16ZXN

6zxn

A1
26ZXN

6zxn

B1
36ZXN

6zxn

C1
43TPK

3tpk

D1
53TPK

3tpk

E1
Refine LS restraints
Refine-IDTypeDev idealNumberWeight
ELECTRON MICROSCOPYf_bond_d0.022278331.431
ELECTRON MICROSCOPYf_angle_d3.238378521.705
ELECTRON MICROSCOPYf_dihedral_angle_d10.6793923
ELECTRON MICROSCOPYf_chiral_restr0.4274383
ELECTRON MICROSCOPYf_plane_restr0.0254852

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