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- PDB-7on5: Crystal structure of the SARS-CoV-2 neutralizing nanobody Re5D06 -

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Basic information

Entry
Database: PDB / ID: 7on5
TitleCrystal structure of the SARS-CoV-2 neutralizing nanobody Re5D06
ComponentsNanobody Re5D06
KeywordsIMMUNE SYSTEM / Neutralizing VHH
Function / homologyETHANOL
Function and homology information
Biological speciesVicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.25 Å
AuthorsAksu, M. / Guttler, T. / Gorlich, D.
CitationJournal: EMBO J / Year: 2021
Title: Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.
Authors: Thomas Güttler / Metin Aksu / Antje Dickmanns / Kim M Stegmann / Kathrin Gregor / Renate Rees / Waltraud Taxer / Oleh Rymarenko / Jürgen Schünemann / Christian Dienemann / Philip Gunkel / ...Authors: Thomas Güttler / Metin Aksu / Antje Dickmanns / Kim M Stegmann / Kathrin Gregor / Renate Rees / Waltraud Taxer / Oleh Rymarenko / Jürgen Schünemann / Christian Dienemann / Philip Gunkel / Bianka Mussil / Jens Krull / Ulrike Teichmann / Uwe Groß / Volker C Cordes / Matthias Dobbelstein / Dirk Görlich /
Abstract: Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) ...Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.
History
DepositionMay 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc
Item: _citation.journal_volume / _citation_author.identifier_ORCID
Revision 1.2Jan 31, 2024Group: 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 Re5D06
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,4633
Polymers14,3551
Non-polymers1082
Water3,675204
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area400 Å2
ΔGint8 kcal/mol
Surface area6990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.611, 44.611, 144.155
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-458-

HOH

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Components

#1: Antibody Nanobody Re5D06


Mass: 14354.760 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Komagataella pastoris (fungus)
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C2H6O2
#3: Chemical ChemComp-EOH / ETHANOL


Mass: 46.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 204 / 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: 2.5 Å3/Da / Density % sol: 50.76 %
Crystal growTemperature: 291 K / Method: vapor diffusion / pH: 6
Details: 100 mM MES: pH 6.0, 200 mM zinc acetate, 15% (v/v) ethanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Nov 6, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.25→42.62 Å / Num. obs: 41182 / % possible obs: 99.43 % / Redundancy: 23.8 % / CC1/2: 0.999 / Rpim(I) all: 0.01136 / Rrim(I) all: 0.0555 / Rsym value: 0.05427 / Net I/σ(I): 38.44
Reflection shellResolution: 1.25→1.295 Å / Redundancy: 14.8 % / Mean I/σ(I) obs: 7.6 / Num. unique obs: 3846 / CC1/2: 0.973 / Rpim(I) all: 0.1053 / Rrim(I) all: 0.4114 / Rsym value: 0.3971 / % possible all: 95.13

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.27data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7OLZ

7olz


Resolution: 1.25→42.62 Å / SU ML: 0.09 / Cross valid method: THROUGHOUT / σ(F): 1.43 / Phase error: 14.62 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1668 2058 5 %
Rwork0.146 39121 -
obs0.147 41179 99.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 79.42 Å2 / Biso mean: 17.6578 Å2 / Biso min: 7.93 Å2
Refinement stepCycle: final / Resolution: 1.25→42.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms989 0 7 204 1200
Biso mean--27.22 30.57 -
Num. residues----127
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.25-1.280.24121270.19442421254894
1.28-1.310.20731320.15552521265398
1.31-1.350.18391350.138825602695100
1.35-1.390.15991340.1325572691100
1.39-1.430.1671360.126725802716100
1.43-1.480.17281360.123925812717100
1.48-1.540.15011380.116426052743100
1.54-1.610.15371360.114125772713100
1.61-1.70.15241350.118725982733100
1.7-1.80.15491390.132226182757100
1.8-1.940.16571380.138326242762100
1.94-2.140.15311380.134126372775100
2.14-2.450.16141400.153526512791100
2.45-3.080.17871420.163427102852100
3.08-42.620.16921520.160228813033100

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