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- PDB-7kl9: Structure of the SARS-CoV-2 S 6P trimer in complex with the ACE2 ... -

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Basic information

Entry
Database: PDB / ID: 7kl9
TitleStructure of the SARS-CoV-2 S 6P trimer in complex with the ACE2 protein decoy, CTC-445.2 (State 4)
Components
  • CTC-445.2 inhibitor
  • Spike glycoproteinPeplomer
KeywordsANTIVIRAL PROTEIN / SARS-CoV-2 / ACE2 decoy / mini-protein / inhibitor / COVID-19
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 / host cell surface receptor binding / endocytosis involved in viral entry into host cell / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host plasma membrane / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host plasma membrane / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Spike receptor binding domain, betacoronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike glycoprotein S2, coronavirus / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S1, C-terminal
Spike glycoprotein / polysac:dglcpnacb1-4dglcpnacb1-roh:
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsBarnes, C.O. / Bjorkman, P.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01-AI13893 United States
CitationJournal: Science / Year: 2020
Title: De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.
Authors: Thomas W Linsky / Renan Vergara / Nuria Codina / Jorgen W Nelson / Matthew J Walker / Wen Su / Christopher O Barnes / Tien-Ying Hsiang / Katharina Esser-Nobis / Kevin Yu / Z Beau Reneer / ...Authors: Thomas W Linsky / Renan Vergara / Nuria Codina / Jorgen W Nelson / Matthew J Walker / Wen Su / Christopher O Barnes / Tien-Ying Hsiang / Katharina Esser-Nobis / Kevin Yu / Z Beau Reneer / Yixuan J Hou / Tanu Priya / Masaya Mitsumoto / Avery Pong / Uland Y Lau / Marsha L Mason / Jerry Chen / Alex Chen / Tania Berrocal / Hong Peng / Nicole S Clairmont / Javier Castellanos / Yu-Ru Lin / Anna Josephson-Day / Ralph S Baric / Deborah H Fuller / Carl D Walkey / Ted M Ross / Ryan Swanson / Pamela J Bjorkman / Michael Gale / Luis M Blancas-Mejia / Hui-Ling Yen / Daniel-Adriano Silva /
Abstract: We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, ...We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
E: CTC-445.2 inhibitor
D: CTC-445.2 inhibitor
F: CTC-445.2 inhibitor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)476,48835
Polymers469,6676
Non-polymers6,82129
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area35170 Å2
ΔGint-60 kcal/mol
Surface area155370 Å2

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Components

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


Mass: 139339.312 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 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Protein CTC-445.2 inhibitor


Mass: 17216.268 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-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.: 27 / 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
1Ternary complex of CTC445.2 inhibitor with SARS-CoV-2 S 6P glycoprotinCOMPLEX#10RECOMBINANT
2Spike glycoproteinPeplomerCOMPLEX1RECOMBINANT
3CTC-445.2 inhibitorCOMPLEX1RECOMBINANT
Molecular weightValue: .6 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
23Homo sapiens (human)9606
12Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
10.02 MTrisTris-HCLTris1
20.12 MSodium ChlorideNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportDetails: 0.2 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 3s blot, 0 blot force

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA / Details: 3x3 beam tilt collection
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.6 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2250

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
12cryoSPARCv2.14classification
13cryoSPARCv2.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 420998
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26038 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00826887
ELECTRON MICROSCOPYf_angle_d0.82736604
ELECTRON MICROSCOPYf_dihedral_angle_d12.5473876
ELECTRON MICROSCOPYf_chiral_restr0.0514275
ELECTRON MICROSCOPYf_plane_restr0.0054755

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