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- PDB-8q94: Crystal structure of The SARS-COV-2 BA.2.75 RBD with neutralizing... -

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Basic information

Entry
Database: PDB / ID: 8q94
TitleCrystal structure of The SARS-COV-2 BA.2.75 RBD with neutralizing-VHHs Re32D03 and Ma3B12
Components
  • Nanobody Ma3B12
  • Nanobody Re32D03
  • Spike protein S1
KeywordsVIRAL PROTEIN / Neutralizing VHH / Fold-promoter
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
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsAksu, M. / Guttler, T. / 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

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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 Ma3B12
C: Nanobody Re32D03


Theoretical massNumber of molelcules
Total (without water)49,7273
Polymers49,7273
Non-polymers00
Water50428
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3180 Å2
ΔGint-17 kcal/mol
Surface area19490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.159, 67.159, 224.897
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Spike protein S1


Mass: 21316.883 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 Ma3B12


Mass: 14308.659 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 Re32D03


Mass: 14101.843 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)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 28 / Source method: isolated from a natural source / Formula: H2O

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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: 2.56 Å3/Da / Density % sol: 51.89 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 7 / Details: 0.1 M HEPES pH 7.0, 18% PEG 12000

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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: Sep 9, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.5→47.49 Å / Num. obs: 485338 / % possible obs: 99.84 % / Redundancy: 26 % / CC1/2: 0.999 / Rpim(I) all: 0.02947 / Rrim(I) all: 0.1503 / Net I/σ(I): 21.15
Reflection shellResolution: 2.5→2.589 Å / Redundancy: 27.4 % / Mean I/σ(I) obs: 3.13 / Num. unique obs: 1809 / CC1/2: 0.992 / Rpim(I) all: 0.3162 / Rrim(I) all: 1.672 / % possible all: 99.83

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Processing

Software
NameVersionClassification
PHENIX1.20.1refinement
pointlessdata scaling
XDSdata reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→47.49 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 31.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.277 933 5 %
Rwork0.2195 --
obs0.2224 18675 99.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.5→47.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3305 0 0 28 3333
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023396
X-RAY DIFFRACTIONf_angle_d0.4614621
X-RAY DIFFRACTIONf_dihedral_angle_d9.9491199
X-RAY DIFFRACTIONf_chiral_restr0.042486
X-RAY DIFFRACTIONf_plane_restr0.003603
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.630.35761270.28492435X-RAY DIFFRACTION100
2.63-2.80.29951320.28072500X-RAY DIFFRACTION100
2.8-3.010.35961290.28162467X-RAY DIFFRACTION100
3.01-3.320.33951310.2512495X-RAY DIFFRACTION100
3.32-3.80.311330.23412535X-RAY DIFFRACTION100
3.8-4.780.21171360.17992574X-RAY DIFFRACTION100
4.78-47.490.25621450.1982736X-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)
12.5144-1.08222.45536.9289-2.60942.7667-0.6972-0.33830.30631.10460.5396-0.4954-1.25730.23650.17050.61560.07460.03730.47580.0150.425631.7178-41.2915-11.7369
24.92072.42914.57723.65680.52865.5783-0.3421.21920.1995-0.37310.3786-0.0377-0.45960.5489-0.01580.3323-0.07330.12350.4003-0.01560.42234.2034-46.3674-22.1953
37.3870.2067-1.14024.6282-1.21772.8564-0.24070.02330.11740.20720.1657-0.38660.17440.48750.12380.49-0.04020.04660.41050.00910.367634.5503-50.952-12.8331
46.25384.64613.2965.65566.19968.12190.45160.70070.20450.1233-0.33230.3271.21170.2836-0.31970.52170.0279-0.11480.46330.0230.442323.9649-41.8977-23.0945
59.9047-2.40392.42512.9948-2.27575.86520.4053-0.15-0.48890.0962-0.29180.230.5511-0.3742-0.14920.5517-0.0119-0.06260.3292-0.05790.309929.0734-45.1851-7.2859
66.6084-1.2838-0.28741.95361.35248.6985-0.10010.3096-0.01470.02350.12040.3445-0.0607-1.25010.00480.5182-0.0290.00650.62560.09910.4868-7.9802-29.1545-22.1362
72.6951-0.6317-0.7661.3239-0.0071.2877-0.03480.13110.07790.02220.07590.051-0.1175-0.2157-0.03040.5767-0.0442-0.0360.49880.01960.36789.115-32.9162-19.7351
85.2221.7022-0.43834.2024-0.38311.51420.2099-0.35360.13170.4684-0.32490.0113-0.3969-0.12620.09440.45440.0918-0.01280.59760.01470.3718-2.5464-29.38429.4954
93.1089-1.57753.51075.9304-2.70724.38170.2608-0.1791-0.4663-0.576-0.11670.64720.1718-0.5043-0.06340.57030.08630.17790.7770.03640.399-8.49-33.03211.0941
100.9966-1.59360.24794.0620.99864.03080.1616-0.10030.28790.4883-0.0450.5181-0.0017-1.0951-0.07110.5019-0.05480.1020.67040.01350.4235-13.6663-32.87347.9925
112.8144-0.01010.19042.65760.35190.67490.07410.0181-0.0137-0.0357-0.02210.0602-0.1682-0.2703-0.00550.59060.00540.0220.64090.0210.4455-4.9698-33.17775.4722
123.9635-0.20942.2532.8546-0.93111.5109-0.0504-0.72670.07430.1506-0.0098-0.13610.1165-0.2440.02880.6588-0.0087-0.06730.5107-0.03990.345534.32-53.0592-5.7992
135.9832-0.57321.60145.7334-1.57871.49780.0148-0.2019-0.11890.6015-0.04720.2407-0.0613-0.18560.04040.39830.02030.06670.4564-0.01640.305125.9861-51.4073-13.9844
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'C' and (resid 40 through 52 )
2X-RAY DIFFRACTION2chain 'C' and (resid 53 through 67 )
3X-RAY DIFFRACTION3chain 'C' and (resid 68 through 100 )
4X-RAY DIFFRACTION4chain 'C' and (resid 101 through 110 )
5X-RAY DIFFRACTION5chain 'C' and (resid 111 through 127 )
6X-RAY DIFFRACTION6chain 'A' and (resid 336 through 393 )
7X-RAY DIFFRACTION7chain 'A' and (resid 394 through 517 )
8X-RAY DIFFRACTION8chain 'B' and (resid 1 through 39 )
9X-RAY DIFFRACTION9chain 'B' and (resid 40 through 60 )
10X-RAY DIFFRACTION10chain 'B' and (resid 61 through 73 )
11X-RAY DIFFRACTION11chain 'B' and (resid 74 through 128 )
12X-RAY DIFFRACTION12chain 'C' and (resid 1 through 17 )
13X-RAY DIFFRACTION13chain 'C' and (resid 18 through 39 )

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