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

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Basic information

Entry
Database: PDB / ID: 8elo
TitleCrystal structure of SARS-CoV-2 spike protein receptor-binding domain in complex with antibody CC12.1 Fab and nanobody Nb-C4-225
Components
  • CC12.1 Fab heavy chain
  • CC12.1 Fab light chain
  • Nanobody Nb-C4-225
  • 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


symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / viral translation / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion ...symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / viral translation / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / membrane fusion / entry receptor-mediated virion attachment to host cell / Attachment and Entry / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont entry into host cell / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, 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 superfamily, coronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.72 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
Revision 1.2Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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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-C4-225
H: CC12.1 Fab heavy chain
L: CC12.1 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,5245
Polymers85,3034
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)88.674, 143.052, 147.725
Angle α, β, γ (deg.)90.000, 90.000, 90.000
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-C4-225


Mass: 15377.755 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
Has protein modificationY

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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: 2.75 Å3/Da / Density % sol: 55.21 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 20% (w/v) PEG-3350, 0.2 M di-Ammonium citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-1 / Wavelength: 0.97946 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 22, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 24814 / % possible obs: 96.9 % / Redundancy: 4.1 % / Biso Wilson estimate: 47.67 Å2 / Rmerge(I) obs: 0.117 / Rpim(I) all: 0.061 / Rrim(I) all: 0.133 / Χ2: 0.828 / Net I/σ(I): 8.4 / Num. measured all: 102906
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.7-2.754.11.24212210.4670.6431.4070.83597.5
2.75-2.840.99312230.5220.5261.1310.82196.8
2.8-2.853.80.81111980.5480.4310.9250.83893.8
2.85-2.913.90.71312100.6450.3720.8090.86797.3
2.91-2.974.30.58612670.7880.2990.6610.87599.2
2.97-3.044.40.46512520.8410.2360.5240.9399.4
3.04-3.124.30.3712680.8920.1880.4170.87699.3
3.12-3.24.30.30512360.9180.1570.3450.90798.8
3.2-3.34.30.24712570.940.1270.2790.95298.7
3.3-3.44.20.19512590.9570.1020.2210.94599
3.4-3.524.20.15912490.9660.0840.1810.9598.5
3.52-3.664.20.13112520.9740.0690.1490.88398.3
3.66-3.834.10.10712540.9830.0570.1220.87197.4
3.83-4.0340.09112260.9830.0490.1040.80396.3
4.03-4.293.70.07411800.9850.0410.0850.70292.1
4.29-4.624.30.07312480.9870.0380.0830.77197
4.62-5.084.30.0712410.9880.0360.0790.71596.6
5.08-5.814.30.06912640.9870.0350.0780.69396.6
5.81-7.324.10.06412480.9860.0340.0730.67894.8
7.32-503.80.05412610.9920.030.0630.57690.6

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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: 2.72→38.01 Å / SU ML: 0.39 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 27.87 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2612 1215 5.22 %
Rwork0.22 22063 -
obs0.2222 23278 90.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 160.22 Å2 / Biso mean: 54.577 Å2 / Biso min: 20.09 Å2
Refinement stepCycle: final / Resolution: 2.72→38.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5641 0 14 0 5655
Biso mean--77.05 --
Num. residues----749
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 9

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.72-2.830.3781690.29391549161858
2.83-2.950.33541270.28492189231682
2.95-3.110.35891390.27642587272697
3.11-3.310.2821480.25252627277599
3.31-3.560.29991500.23182638278899
3.56-3.920.26591540.22152621277598
3.92-4.480.26561530.18332563271694
4.48-5.650.17811260.18082648277496
5.65-38.010.22471490.21922641279093
Refinement TLS params.Method: refined / Origin x: 6.9817 Å / Origin y: 37.5754 Å / Origin z: 0.7196 Å
111213212223313233
T0.1993 Å20.0111 Å2-0.0525 Å2-0.2814 Å2-0.0398 Å2--0.2713 Å2
L0.4836 °20.3207 °2-0.2593 °2-1.3857 °2-0.5532 °2--0.5693 °2
S-0.0345 Å °0.0239 Å °0.0724 Å °-0.0162 Å °0.0885 Å °0.0586 Å °-0.0249 Å °0.0231 Å °-0.0447 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA334 - 528
2X-RAY DIFFRACTION1allB1 - 112
3X-RAY DIFFRACTION1allH1 - 214
4X-RAY DIFFRACTION1allL1 - 213

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