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- PDB-8elp: Crystal structure of SARS-CoV-2 spike protein receptor-binding do... -

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Basic information

Entry
Database: PDB / ID: 8elp
TitleCrystal structure of SARS-CoV-2 spike protein receptor-binding domain in complex with antibody CC12.1 Fab and nanobody Nb-C4-240
Components
  • CC12.1 Fab heavy chain
  • CC12.1 Fab light chain
  • Nanobody Nb-C4-240
  • Spike protein S1
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / synthetic / nanobody / SARS-CoV-2 / coronavirus / neutralization / IMMUNE SYSTEM / VIRAL PROTEIN-IMMUNE SYSTEM complex
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 / 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
synthetic construct (others)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.83 Å
AuthorsLiu, H. / Wilson, I.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationINV-004923 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.
Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine /
Abstract: Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple ...Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V and V domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
History
DepositionSep 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 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

Downloads & links

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Assembly

Deposited unit
A: Spike protein S1
B: Nanobody Nb-C4-240
H: CC12.1 Fab heavy chain
L: CC12.1 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,7915
Polymers85,5704
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
H: CC12.1 Fab heavy chain
L: CC12.1 Fab light chain


Theoretical massNumber of molelcules
Total (without water)46,8202
Polymers46,8202
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3430 Å2
ΔGint-20 kcal/mol
Surface area19440 Å2
MethodPISA
Unit cell
Length a, b, c (Å)90.222, 144.731, 148.116
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Spike protein S1


Mass: 23104.867 Da / Num. of mol.: 1 / Fragment: Receptor binding domain, UNP residues 333-530
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P0DTC2
#2: Antibody Nanobody Nb-C4-240


Mass: 15645.075 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Homo sapiens (human)
#3: Antibody CC12.1 Fab heavy chain


Mass: 23178.928 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Mus musculus (house mouse)
#4: Antibody CC12.1 Fab light chain


Mass: 23641.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Mus musculus (house mouse)
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
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: 2.83 Å3/Da / Density % sol: 56.46 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 0.16 M ammonium sulfate, 0.08 M sodium acetate pH 4.6, 20% (w/v) polyethylene glycol 4000, 20% (v/v) glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 5, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2.83→50 Å / Num. obs: 42588 / % possible obs: 96.8 % / Redundancy: 2.1 % / Rmerge(I) obs: 0.955 / Χ2: 0.141 / Net I/σ(I): 4.7
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID% possible all
2.85-2.921.90.70926750.49193.2
2.92-2.991.90.68627900.485195.3
2.99-3.0720.57728700.495197.5
3.07-3.162.10.52128770.511197.9
3.16-3.262.10.47228720.533198.1
3.26-3.382.10.34628530.552196.8
3.38-3.511.90.27727770.715194.5
3.51-3.672.20.21928860.693198.6
3.67-3.872.20.17528860.747198.3
3.87-4.112.20.14229090.89198
4.11-4.432.10.10728151.07197
4.43-4.872.10.0828111.133195.8
4.87-5.582.30.07328950.886198
5.58-7.022.10.06828200.742195.9
7.02-502.20.04828521.06196.7

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIX1.19.2_4158refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7KN5, 6XC3

7kn5

6xc3


Resolution: 2.83→43.15 Å / SU ML: 0.41 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 27.75 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2838 1198 5.3 %
Rwork0.2381 --
obs0.2406 22625 96.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.83→43.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5710 0 14 0 5724
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0025865
X-RAY DIFFRACTIONf_angle_d0.537981
X-RAY DIFFRACTIONf_dihedral_angle_d19.229824
X-RAY DIFFRACTIONf_chiral_restr0.042879
X-RAY DIFFRACTIONf_plane_restr0.0041027
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.83-2.950.39031160.3422057X-RAY DIFFRACTION85
2.95-3.080.37561440.30212382X-RAY DIFFRACTION99
3.08-3.240.3261280.26952410X-RAY DIFFRACTION99
3.24-3.450.31721150.27592409X-RAY DIFFRACTION98
3.45-3.710.26151360.23882432X-RAY DIFFRACTION99
3.71-4.090.32451590.22262386X-RAY DIFFRACTION99
4.09-4.680.21581250.19112399X-RAY DIFFRACTION97
4.68-5.890.23441320.1982462X-RAY DIFFRACTION99
5.89-43.150.26581430.23832490X-RAY DIFFRACTION96
Refinement TLS params.Method: refined / Origin x: 7.5947 Å / Origin y: 38.1285 Å / Origin z: 0.8468 Å
111213212223313233
T0.1385 Å20.0026 Å2-0.0206 Å2-0.268 Å2-0.0133 Å2--0.2445 Å2
L0.2132 °20.2968 °2-0.2396 °2-1.2288 °2-0.6549 °2--0.5688 °2
S-0.0025 Å °-0.0717 Å °0.0296 Å °0.0641 Å °0.0055 Å °0.0465 Å °-0.0236 Å °0.025 Å °0.0096 Å °
Refinement TLS groupSelection details: all

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