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- PDB-7a29: Cryo-EM structure of the SARS-CoV-2 spike protein bound to neutra... -

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Basic information

Entry
Database: PDB / ID: 7a29
TitleCryo-EM structure of the SARS-CoV-2 spike protein bound to neutralizing sybodies (Sb23) 2-up conformation
Components
  • Neutralising sybody (Sb23)
  • Spike glycoproteinPeplomer
KeywordsVIRAL PROTEIN / SARS-CoV-2 / complex / nanobody / spike
Function / homology
Function and homology information


suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / viral translation / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / viral protein processing ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / viral translation / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / viral protein processing / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / go:0009405: / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Betacoronavirus spike glycoprotein S1, receptor binding / 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 / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein, betacoronavirus / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / in:ipr027400: / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus
Spike glycoprotein / polysac:dglcpnacb1-4dglcpnacb1-:
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsHallberg, B.M. / Das, H.
CitationJournal: Nat Commun / Year: 2020
Title: Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.
Authors: Tânia F Custódio / Hrishikesh Das / Daniel J Sheward / Leo Hanke / Samuel Pazicky / Joanna Pieprzyk / Michèle Sorgenfrei / Martin A Schroer / Andrey Yu Gruzinov / Cy M Jeffries / Melissa ...Authors: Tânia F Custódio / Hrishikesh Das / Daniel J Sheward / Leo Hanke / Samuel Pazicky / Joanna Pieprzyk / Michèle Sorgenfrei / Martin A Schroer / Andrey Yu Gruzinov / Cy M Jeffries / Melissa A Graewert / Dmitri I Svergun / Nikolay Dobrev / Kim Remans / Markus A Seeger / Gerald M McInerney / Ben Murrell / B Martin Hällberg / Christian Löw /
Abstract: The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional ...The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI
Revision 1.2Nov 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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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
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
D: Neutralising sybody (Sb23)
E: Neutralising sybody (Sb23)
F: Neutralising sybody (Sb23)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)478,14659
Polymers464,7976
Non-polymers13,35053
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / Peplomer / 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 Neutralising sybody (Sb23)


Mass: 12532.897 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)
#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.: 8 / Source method: obtained synthetically
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: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 45 / 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: 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 glycoprotein in 1-up conformation bound by 3 neutralising Sybodies (Sb23)COMPLEX#1-#20MULTIPLE SOURCES
2Spike glycoproteinPeplomerCOMPLEX#11RECOMBINANT
3Neutralising sybody (Sb23)COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Severe acute respiratory syndrome coronavirus 22697049
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo Sapiens (human)9606
23Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1100 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
2EPU2.7image acquisition
13cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69567 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 114.93 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001928911
ELECTRON MICROSCOPYf_angle_d0.516139314
ELECTRON MICROSCOPYf_chiral_restr0.04794602
ELECTRON MICROSCOPYf_plane_restr0.00335003
ELECTRON MICROSCOPYf_dihedral_angle_d11.27564130

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