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- PDB-7olz: Crystal structure of the SARS-CoV-2 RBD with neutralizing-VHHs Re... -

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Basic information

Entry
Database: PDB / ID: 7olz
TitleCrystal structure of the SARS-CoV-2 RBD with neutralizing-VHHs Re5D06 and Re9F06
Components
  • Nanobody Re5D06
  • Nanobody Re9F06
  • Spike protein S1
KeywordsVIRAL PROTEIN / COVID19 / Neutralizing VHH
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
3-[BENZYL(DIMETHYL)AMMONIO]PROPANE-1-SULFONATE / Spike glycoprotein
Similarity search - Component
Biological speciesVicugna pacos (alpaca)
Severe acute respiratory syndrome coronavirus 2
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 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 20, 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

Downloads & links

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Assembly

Deposited unit
C: Nanobody Re5D06
B: Nanobody Re9F06
A: Spike protein S1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,4495
Polymers50,0953
Non-polymers3532
Water4,954275
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3890 Å2
ΔGint-18 kcal/mol
Surface area19350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.955, 57.017, 144.029
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 1 molecules A

#3: Protein Spike protein S1


Mass: 21873.496 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

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Antibody , 2 types, 2 molecules CB

#1: Antibody Nanobody Re5D06


Mass: 14413.827 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)
#2: Antibody Nanobody Re9F06


Mass: 13808.122 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)

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Non-polymers , 3 types, 277 molecules

#4: Chemical ChemComp-DMX / 3-[BENZYL(DIMETHYL)AMMONIO]PROPANE-1-SULFONATE


Mass: 257.349 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H19NO3S
#5: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: SO4
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 275 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.23 Å3/Da / Density % sol: 44.73 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M MOPS pH 7.5, 2.07 M ammonium sulfate, 0.1 M NDSB-256

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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: Oct 24, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.75→53.01 Å / Num. obs: 45701 / % possible obs: 85.05 % / Redundancy: 13.3 % / Biso Wilson estimate: 15.93 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.09045 / Rpim(I) all: 0.026 / Rrim(I) all: 0.094 / Net I/σ(I): 17.91
Reflection shellResolution: 1.75→1.813 Å / Redundancy: 13 % / Rmerge(I) obs: 1.53 / Mean I/σ(I) obs: 1.99 / Num. unique obs: 2363 / CC1/2: 0.873 / Rpim(I) all: 0.43 / Rrim(I) all: 1.59 / % possible all: 51.39

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6YZ5

6yz5


Resolution: 1.75→53.01 Å / SU ML: 0.16 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 21.75 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2092 2048 5.17 %
Rwork0.1757 37544 -
obs0.1775 39592 85.05 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 87.7 Å2 / Biso mean: 23.4695 Å2 / Biso min: 4.94 Å2
Refinement stepCycle: final / Resolution: 1.75→53.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3258 0 39 275 3572
Biso mean--28.15 30.22 -
Num. residues----422
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 (%)
1.75-1.790.2366810.20651465154650
1.79-1.840.2597880.1951608169655
1.84-1.890.2258920.19311835192763
1.89-1.940.26171160.18272037215371
1.94-20.2880.17342291237977
2-2.070.21981340.17542410254483
2.07-2.160.22421330.17492546267988
2.16-2.260.21311490.17662740288993
2.26-2.370.21471550.17122867302298
2.38-2.520.21751530.17842889304299
2.52-2.720.21911580.1812898305698
2.72-2.990.21281760.1822903307999
2.99-3.430.21081680.17452961312999
3.43-4.310.18241750.15512961313699
4.32-53.010.19791820.18363133331599
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)
10.9319-0.2949-0.26092.9889-0.75671.23150.12770.01750.19630.0799-0.02430.1424-0.31560.1987-0.03660.1636-0.05330.04970.1254-0.02160.1331-6.99772.6088-7.5895
22.0764-0.23390.73322.30860.03762.2513-0.05640.0327-0.0994-0.1370.09560.46540.0566-0.1222-0.01890.1434-0.0212-0.0260.10740.01220.148-5.5955-27.3925-40.1293
31.74830.19170.38991.3470.61442.0036-0.0130.1131-0.048-0.03290.0476-0.11080.1110.20280.00420.05090.00020.00830.0937-0.01230.064610.7374-22.13-15.5338
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'C' and resid 1 through 129)C1 - 129
2X-RAY DIFFRACTION2(chain 'B' and resid 1 through 122)B1 - 122
3X-RAY DIFFRACTION3(chain 'A' and resid 335 through 515)A335 - 515

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