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- PDB-7p78: SARS-CoV-2 spike protein in complex with sybody#15 and sybody#68 ... -

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Basic information

Entry
Database: PDB / ID: 7p78
TitleSARS-CoV-2 spike protein in complex with sybody#15 and sybody#68 in a 1up/1up-out/1down conformation
Components
  • Spike glycoprotein
  • sybody#15
  • sybody#68
KeywordsVIRAL PROTEIN / SARS-CoV-2 spike protein sybody
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 / entry receptor-mediated virion attachment to host cell ...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 / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / membrane fusion / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / 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 S1 subunit, receptor binding domain superfamily, 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, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / 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 S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike glycoprotein S1, receptor binding / 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 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsWalter, J.D. / Hutter, C.A.J. / Garaeva, A.A. / Scherer, M. / Zimmermann, I. / Wyss, M. / Rheinberger, J. / Ruedin, Y. / Earp, J.C. / Egloff, P. ...Walter, J.D. / Hutter, C.A.J. / Garaeva, A.A. / Scherer, M. / Zimmermann, I. / Wyss, M. / Rheinberger, J. / Ruedin, Y. / Earp, J.C. / Egloff, P. / Sorgenfrei, M. / Huerlimann, L.M. / Gonda, I. / Meier, G. / Remm, S. / Thavarasah, S. / Zimmer, G. / Slotboom, D.J. / Paulino, C. / Plattet, P. / Seeger, M.A.
Funding support Netherlands, Switzerland, 3items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
Swiss National Science Foundation4078P0_198314 Switzerland
Citation
Journal: EMBO Rep / Year: 2022
Title: Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.
Authors: Justin D Walter / Melanie Scherer / Cedric A J Hutter / Alisa A Garaeva / Iwan Zimmermann / Marianne Wyss / Jan Rheinberger / Yelena Ruedin / Jennifer C Earp / Pascal Egloff / Michèle ...Authors: Justin D Walter / Melanie Scherer / Cedric A J Hutter / Alisa A Garaeva / Iwan Zimmermann / Marianne Wyss / Jan Rheinberger / Yelena Ruedin / Jennifer C Earp / Pascal Egloff / Michèle Sorgenfrei / Lea M Hürlimann / Imre Gonda / Gianmarco Meier / Sille Remm / Sujani Thavarasah / Geert van Geest / Rémy Bruggmann / Gert Zimmer / Dirk J Slotboom / Cristina Paulino / Philippe Plattet / Markus A Seeger /
Abstract: The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously ...The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.
#1: Journal: bioRxiv / Year: 2020
Title: Bispecific sybody constructs neutralize SARS-CoV-2 variants of concern and mitigate emergence of drug-resistance
Authors: Walter, J.D. / Hutter, C.A.J. / Garaeva, A.A. / Scherer, M. / Zimmermann, I. / Wyss, M. / Rheinberger, J. / Ruedin, Y. / Earp, J.C. / Egloff, P. / Sorgenfrei, M. / Huerlimann, L.M. / Gonda, ...Authors: Walter, J.D. / Hutter, C.A.J. / Garaeva, A.A. / Scherer, M. / Zimmermann, I. / Wyss, M. / Rheinberger, J. / Ruedin, Y. / Earp, J.C. / Egloff, P. / Sorgenfrei, M. / Huerlimann, L.M. / Gonda, I. / Meier, G. / Remm, S. / Thavarasah, S. / Zimmer, G. / Slotboom, D.J. / Paulino, C. / Plattet, P. / Seeger, M.A.
History
DepositionJul 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_entity_branch_descriptor
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Apr 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / em_3d_fitting_list / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _citation.journal_id_ISSN / _em_3d_fitting_list.accession_code ..._citation.journal_id_ISSN / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
C: Spike glycoprotein
E: Spike glycoprotein
A: Spike glycoprotein
B: sybody#15
D: sybody#15
H: sybody#15
W: sybody#68
Y: sybody#68
hetero molecules


Theoretical massNumber of molelcules
Total (without water)503,52843
Polymers491,6838
Non-polymers11,84535
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44170 Å2
ΔGint-23 kcal/mol
Surface area167870 Å2
MethodPISA

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Components

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Protein , 2 types, 6 molecules CEABDH

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 142399.375 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Protein sybody#15


Mass: 12531.054 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Antibody , 1 types, 2 molecules WY

#3: Antibody sybody#68


Mass: 13445.861 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Sugars , 4 types, 35 molecules

#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(3+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(5-4)-2- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(5-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 627.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb5-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+2)][a-L-Sorp5NAc]{[(3+1)][b-D-GlcpNAc]{}}}}LINUCSPDB-CARE
#7: 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.: 19 / 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

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SARS-CoV-2 spike protein in complex with sybody#15 and sybody#68COMPLEX#2-#30MULTIPLE SOURCES
2SARS-CoV-2 spike proteinCOMPLEX#11RECOMBINANT
3sybody#15 and sybody#68COMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Severe acute respiratory syndrome coronavirus 22697049
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
Buffer solutionpH: 8 / Details: 2 mM Tris-HCl pH 8.0, 200 mM NaCl
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 12454
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisitioncollected with a 3x3 pattern
4CTFFIND4.1.13CTF correction
7Coot0.9-premodel fitting
8UCSF Chimera0.93model fittingChimeraX
10RELION3.0.8initial Euler assignment
11cryoSPARC2.15.0final Euler assignment
13cryoSPARC2.15.03D reconstruction
14PHENIX1.18.2-3874-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 637105
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52839 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17MY2

7my2

17MY21PDBexperimental model
23K1K

3k1k

13K1K2PDBexperimental model
37KLW

7klw

17KLW3PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00629447
ELECTRON MICROSCOPYf_angle_d1.0840073
ELECTRON MICROSCOPYf_dihedral_angle_d13.71710477
ELECTRON MICROSCOPYf_chiral_restr0.0654699
ELECTRON MICROSCOPYf_plane_restr0.0095093

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