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- PDB-7p7a: SARS-CoV-2 spike protein in complex with sybody#68 in a 2up/1flex... -

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Basic information

Entry
Database: PDB / ID: 7p7a
TitleSARS-CoV-2 spike protein in complex with sybody#68 in a 2up/1flexible conformation
Components
  • Spike glycoprotein
  • 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: 4.76 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-12085
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
B: Spike glycoprotein
C: Spike glycoprotein
E: Spike glycoprotein
A: sybody#68
D: sybody#68
hetero molecules


Theoretical massNumber of molelcules
Total (without water)467,96949
Polymers454,1745
Non-polymers13,79544
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38730 Å2
ΔGint37 kcal/mol
Surface area155040 Å2
MethodPISA

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Components

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Protein / Antibody , 2 types, 5 molecules BCEAD

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


Mass: 142427.438 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: 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 , 5 types, 44 molecules

#3: 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.: 10
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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#4: 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
#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 383.349 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-2/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(3+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.: 28 / 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

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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#68COMPLEX#1-#20MULTIPLE SOURCES
2SARS-CoV-2 spike proteinCOMPLEX#11RECOMBINANT
3sybody#68COMPLEX#21RECOMBINANT
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.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 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: 1 / Num. of real images: 5109
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
1crYOLO1.7.5particle selection
2SerialEMimage acquisitioncollected with a 3x3 pattern
4CTFFIND4.1.13CTF correction
7Coot0.9-premodel fitting
8UCSF Chimera0.93model fittingChimeraX
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
14PHENIX1.18.2-3874-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 344976
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24325 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
17MY2

7my2

1
27KLW

7klw

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00725992
ELECTRON MICROSCOPYf_angle_d1.1935400
ELECTRON MICROSCOPYf_dihedral_angle_d13.7999332
ELECTRON MICROSCOPYf_chiral_restr0.0674215
ELECTRON MICROSCOPYf_plane_restr0.014486

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