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- PDB-7p7b: SARS-CoV-2 spike protein in complex with sybody no68 in a 1up/2do... -

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Basic information

Entry
Database: PDB / ID: 7p7b
TitleSARS-CoV-2 spike protein in complex with sybody no68 in a 1up/2down conformation
ComponentsSpike glycoprotein
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 / 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 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: Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / database_2 / struct
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct.title
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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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein


Theoretical massNumber of molelcules
Total (without water)427,2823
Polymers427,2823
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28440 Å2
ΔGint-142 kcal/mol
Surface area140850 Å2

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Components

#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

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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

ComponentName: SARS-CoV-2 spike protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human)
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 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
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: 344976
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84917 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6ZGG

6zgg

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00625684
ELECTRON MICROSCOPYf_angle_d1.00534959
ELECTRON MICROSCOPYf_dihedral_angle_d10.4549212
ELECTRON MICROSCOPYf_chiral_restr0.0583953
ELECTRON MICROSCOPYf_plane_restr0.0084545

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