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- PDB-7kkj: Structure of anti-SARS-CoV-2 Spike nanobody mNb6 -

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Entry
Database: PDB / ID: 7kkj
TitleStructure of anti-SARS-CoV-2 Spike nanobody mNb6
ComponentsSynthetic nanobody mNb6
KeywordsIMMUNE SYSTEM / Complex / Nanobody / VHH
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsSchoof, M.S. / Faust, B.F. / Saunders, R.A. / Sangwan, S. / Rezelj, V. / Hoppe, N. / Boone, M. / Billesboelle, C.B. / Puchades, C. / Azumaya, C.M. ...Schoof, M.S. / Faust, B.F. / Saunders, R.A. / Sangwan, S. / Rezelj, V. / Hoppe, N. / Boone, M. / Billesboelle, C.B. / Puchades, C. / Azumaya, C.M. / Kratochvil, H.T. / Zimanyi, M. / Desphande, I. / Liang, J. / Dickinson, S. / Nguyen, H.C. / Chio, C.M. / Merz, G.E. / Thompson, M.C. / Diwanji, D. / Schaefer, K. / Anand, A.A. / Dobzinski, N. / Zha, B.S. / Simoneau, C.R. / Leon, K. / White, K.M. / Chio, U.S. / Gupta, M. / Jin, M. / Li, F. / Liu, Y. / Zhang, K. / Bulkley, D. / Sun, M. / Smith, A.M. / Rizo, A.N. / Moss, F. / Brilot, A.F. / Pourmal, S. / Trenker, R. / Pospiech, T. / Gupta, S. / Barsi-Rhyne, B. / Belyy, V. / Barile-Hill, A.W. / Nock, S. / Liu, Y. / Krogan, N.J. / Ralston, C.Y. / Swaney, D.L. / Garcia-Sastre, A. / Ott, M. / Vignuzzi, M. / Walter, P. / Manglik, A. / QCRG Structural Biology Consortium
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Science / Year: 2020
Title: An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.
Authors: Michael Schoof / Bryan Faust / Reuben A Saunders / Smriti Sangwan / Veronica Rezelj / Nick Hoppe / Morgane Boone / Christian B Billesbølle / Cristina Puchades / Caleigh M Azumaya / Huong T ...Authors: Michael Schoof / Bryan Faust / Reuben A Saunders / Smriti Sangwan / Veronica Rezelj / Nick Hoppe / Morgane Boone / Christian B Billesbølle / Cristina Puchades / Caleigh M Azumaya / Huong T Kratochvil / Marcell Zimanyi / Ishan Deshpande / Jiahao Liang / Sasha Dickinson / Henry C Nguyen / Cynthia M Chio / Gregory E Merz / Michael C Thompson / Devan Diwanji / Kaitlin Schaefer / Aditya A Anand / Niv Dobzinski / Beth Shoshana Zha / Camille R Simoneau / Kristoffer Leon / Kris M White / Un Seng Chio / Meghna Gupta / Mingliang Jin / Fei Li / Yanxin Liu / Kaihua Zhang / David Bulkley / Ming Sun / Amber M Smith / Alexandrea N Rizo / Frank Moss / Axel F Brilot / Sergei Pourmal / Raphael Trenker / Thomas Pospiech / Sayan Gupta / Benjamin Barsi-Rhyne / Vladislav Belyy / Andrew W Barile-Hill / Silke Nock / Yuwei Liu / Nevan J Krogan / Corie Y Ralston / Danielle L Swaney / Adolfo García-Sastre / Melanie Ott / Marco Vignuzzi / / Peter Walter / Aashish Manglik /
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). ...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
History
DepositionOct 27, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 25, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Synthetic nanobody mNb6
C: Synthetic nanobody mNb6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,7027
Polymers27,2822
Non-polymers4205
Water2,360131
1
A: Synthetic nanobody mNb6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,9655
Polymers13,6411
Non-polymers3244
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Synthetic nanobody mNb6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,7372
Polymers13,6411
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)44.569, 71.253, 46.430
Angle α, β, γ (deg.)90.000, 114.930, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Antibody Synthetic nanobody mNb6


Mass: 13641.123 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET26b / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 131 / 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: 2.45 Å3/Da / Density % sol: 49.87 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.1 M Tris-HCl, pH 8.5, 1.0 M Aammonium sulfate

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Data collection

DiffractionMean temperature: 80 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.11 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jul 2, 2020
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.11 Å / Relative weight: 1
ReflectionResolution: 2.05→42.105 Å / Num. obs: 16591 / % possible obs: 97.8 % / Redundancy: 6.4 % / Rmerge(I) obs: 0.13 / Rpim(I) all: 0.05 / Net I/σ(I): 7.2
Reflection shellResolution: 2.05→2.09 Å / Rmerge(I) obs: 0.94 / Mean I/σ(I) obs: 0.9 / Num. unique obs: 1582 / Rpim(I) all: 0.39 / % possible all: 96.6

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Processing

Software
NameVersionClassification
XSCALEdata scaling
BUSTER2.10.3refinement
PDB_EXTRACT3.25data extraction
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5VNV

5vnv


Resolution: 2.05→42.105 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 24.41 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2475 902 5.57 %
Rwork0.2116 --
obs0.2136 16204 97.67 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 93.5 Å2 / Biso mean: 36.1272 Å2 / Biso min: 18.9 Å2
Refinement stepCycle: final / Resolution: 2.05→42.105 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1793 0 21 131 1945
Biso mean--57.58 38.55 -
Num. residues----237

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