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- PDB-7vpy: Crystal structure of the neutralizing nanobody P86 against SARS-CoV-2 -

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Entry
Database: PDB / ID: 7vpy
TitleCrystal structure of the neutralizing nanobody P86 against SARS-CoV-2
ComponentsNanobody
KeywordsIMMUNE SYSTEM / SARS-CoV-2
Biological speciesVicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsMaeda, R. / Fujita, J. / Konishi, Y. / Kazuma, Y. / Yamazaki, H. / Anzai, I. / Yamaguchi, K. / Kasai, K. / Nagata, K. / Yamaoka, Y. ...Maeda, R. / Fujita, J. / Konishi, Y. / Kazuma, Y. / Yamazaki, H. / Anzai, I. / Yamaguchi, K. / Kasai, K. / Nagata, K. / Yamaoka, Y. / Miyakawa, K. / Ryo, A. / Shirakawa, K. / Makino, F. / Matsuura, Y. / Inoue, T. / Imura, A. / Namba, K. / Takaori-Kondo, A.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)20fk0108268 Japan
Japan Agency for Medical Research and Development (AMED)20fk018517 Japan
Japan Agency for Medical Research and Development (AMED)20fk018413 Japan
Japan Science and Technology Japan
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Commun Biol / Year: 2022
Title: A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.
Authors: Ryota Maeda / Junso Fujita / Yoshinobu Konishi / Yasuhiro Kazuma / Hiroyuki Yamazaki / Itsuki Anzai / Tokiko Watanabe / Keishi Yamaguchi / Kazuki Kasai / Kayoko Nagata / Yutaro Yamaoka / Kei ...Authors: Ryota Maeda / Junso Fujita / Yoshinobu Konishi / Yasuhiro Kazuma / Hiroyuki Yamazaki / Itsuki Anzai / Tokiko Watanabe / Keishi Yamaguchi / Kazuki Kasai / Kayoko Nagata / Yutaro Yamaoka / Kei Miyakawa / Akihide Ryo / Kotaro Shirakawa / Kei Sato / Fumiaki Makino / Yoshiharu Matsuura / Tsuyoshi Inoue / Akihiro Imura / Keiichi Namba / Akifumi Takaori-Kondo /
Abstract: We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, ...We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.
History
DepositionOct 18, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 20, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nanobody
B: Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,21612
Polymers31,2892
Non-polymers92710
Water3,927218
1
A: Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,1256
Polymers15,6451
Non-polymers4805
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,0916
Polymers15,6451
Non-polymers4465
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)105.078, 43.166, 56.027
Angle α, β, γ (deg.)90.000, 110.758, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11A-389-

HOH

21B-412-

HOH

31B-417-

HOH

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Components

#1: Antibody Nanobody


Mass: 15644.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 218 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.9 Å3/Da / Density % sol: 35.22 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 51 mg/mL protein, 0.2 M Ammonium sulfate, and 30% w/v polyethylene glycol 4,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 23, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 1.6→49.13 Å / Num. obs: 30851 / % possible obs: 99.1 % / Redundancy: 3.4 % / Biso Wilson estimate: 17.41 Å2 / CC1/2: 0.998 / Rrim(I) all: 0.067 / Net I/σ(I): 11.9
Reflection shellResolution: 1.6→1.63 Å / Mean I/σ(I) obs: 2.8 / Num. unique obs: 1493 / CC1/2: 0.835 / Rrim(I) all: 0.668

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5ivo

5ivo


Resolution: 1.6→30.8 Å / SU ML: 0.1602 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 22.4932
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2131 1510 4.9 %
Rwork0.1769 29324 -
obs0.1787 30834 98.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 23.61 Å2
Refinement stepCycle: LAST / Resolution: 1.6→30.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1770 0 49 218 2037
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00611973
X-RAY DIFFRACTIONf_angle_d0.87192687
X-RAY DIFFRACTIONf_chiral_restr0.0618282
X-RAY DIFFRACTIONf_plane_restr0.0061352
X-RAY DIFFRACTIONf_dihedral_angle_d6.1701284
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.650.25121250.2342646X-RAY DIFFRACTION99
1.65-1.710.23961160.20942670X-RAY DIFFRACTION99.15
1.71-1.780.26661440.20782637X-RAY DIFFRACTION99.04
1.78-1.860.26671500.19272652X-RAY DIFFRACTION98.87
1.86-1.960.18431270.17052673X-RAY DIFFRACTION99.04
1.96-2.080.19811290.17182679X-RAY DIFFRACTION99.5
2.08-2.240.23521550.17362652X-RAY DIFFRACTION99.19
2.24-2.470.22921380.18452650X-RAY DIFFRACTION99.15
2.47-2.820.24231390.1962688X-RAY DIFFRACTION98.71
2.82-3.560.2221360.17282664X-RAY DIFFRACTION97.77
3.56-30.80.17071510.15492713X-RAY DIFFRACTION97.75

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