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- PDB-8q93: Crystal structure of the SARS-COV-2 RBD with neutralizing-VHHs Re... -

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Basic information

Entry
Database: PDB / ID: 8q93
TitleCrystal structure of the SARS-COV-2 RBD with neutralizing-VHHs Re30H02 and Re21D01
Components
  • Nanobody Re21D01
  • Nanobody Re30H02
  • Spike protein S1
KeywordsVIRAL PROTEIN / Neutralizing VHH / Fold-promoter
Function / homology
Function and homology information


symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / viral translation / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion ...symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / viral translation / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / membrane fusion / entry receptor-mediated virion attachment to host cell / Attachment and Entry / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont entry into host cell / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, 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 superfamily, coronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsAksu, M. / Guttler, T. / Rymarenko, O. / Gorlich, D.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Antiviral Res / Year: 2024
Title: Nanobodies to multiple spike variants and inhalation of nanobody-containing aerosols neutralize SARS-CoV-2 in cell culture and hamsters.
Authors: Metin Aksu / Priya Kumar / Thomas Güttler / Waltraud Taxer / Kathrin Gregor / Bianka Mußil / Oleh Rymarenko / Kim M Stegmann / Antje Dickmanns / Sabrina Gerber / Wencke Reineking / Claudia ...Authors: Metin Aksu / Priya Kumar / Thomas Güttler / Waltraud Taxer / Kathrin Gregor / Bianka Mußil / Oleh Rymarenko / Kim M Stegmann / Antje Dickmanns / Sabrina Gerber / Wencke Reineking / Claudia Schulz / Timo Henneck / Ahmed Mohamed / Gerhard Pohlmann / Mehmet Ramazanoglu / Kemal Mese / Uwe Groß / Tamar Ben-Yedidia / Oded Ovadia / Dalit Weinstein Fischer / Merav Kamensky / Amir Reichman / Wolfgang Baumgärtner / Maren von Köckritz-Blickwede / Matthias Dobbelstein / Dirk Görlich /
Abstract: The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single- ...The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.
History
DepositionAug 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike protein S1
B: Nanobody Re21D01
C: Nanobody Re30H02


Theoretical massNumber of molelcules
Total (without water)48,6183
Polymers48,6183
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3750 Å2
ΔGint-17 kcal/mol
Surface area19910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)155.615, 155.615, 155.615
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number207
Space group name H-MP432

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Components

#1: Protein Spike protein S1


Mass: 21949.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Escherichia coli (E. coli) / References: UniProt: P0DTC2
#2: Antibody Nanobody Re21D01


Mass: 12508.881 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#3: Antibody Nanobody Re30H02


Mass: 14159.519 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.23 Å3/Da / Density % sol: 61.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 28% PEG smear broad, 50 mM arginine, 50 mM MSG, 5% Glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Nov 28, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.1→49.62 Å / Num. obs: 12249 / % possible obs: 99.86 % / Redundancy: 76.9 % / CC1/2: 0.997 / Rpim(I) all: 0.04315 / Rrim(I) all: 0.3776 / Rsym value: 0.375 / Net I/σ(I): 20.65
Reflection shellResolution: 3.1→3.21 Å / Mean I/σ(I) obs: 2.36 / Num. unique obs: 1181 / CC1/2: 0.776 / Rpim(I) all: 0.4206 / Rrim(I) all: 3.811 / Rsym value: 3.787

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
pointlessdata scaling
XDSdata reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.1→46.92 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 28.15 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2572 613 5.01 %
Rwork0.2004 --
obs0.2033 12242 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.1→46.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3360 0 0 0 3360
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0053455
X-RAY DIFFRACTIONf_angle_d0.7714702
X-RAY DIFFRACTIONf_dihedral_angle_d15.4221209
X-RAY DIFFRACTIONf_chiral_restr0.05503
X-RAY DIFFRACTIONf_plane_restr0.005614
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.410.32441490.23342820X-RAY DIFFRACTION100
3.41-3.910.27371490.20992846X-RAY DIFFRACTION100
3.91-4.920.23181520.18712888X-RAY DIFFRACTION100
4.93-46.920.25091630.19743075X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.85940.0552-2.17950.54050.2652.8586-0.0809-0.32770.02470.01990.1107-0.03480.15170.0411-0.05150.5260.0107-0.05570.46270.00140.564827.9428-57.0222-39.5108
22.86511.40160.75414.4047-0.19842.4108-0.06880.206-0.2964-0.5610.1440.39950.4206-0.3757-0.09170.6211-0.1087-0.02840.6271-0.02180.579320.149-80.2303-48.4063
36.78450.0738-0.36812.07340.43033.0443-0.2566-0.7703-0.59210.13420.2113-0.0140.114-0.02240.04770.5869-0.00880.01970.72640.10870.5378-3.8184-55.0385-28.2497
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain 'A' and resid 333 through 526)
2X-RAY DIFFRACTION2(chain 'B' and resid 1 through 114)
3X-RAY DIFFRACTION3(chain 'C' and resid 1 through 130)

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