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- PDB-7e7b: Cryo-EM structure of the SARS-CoV-2 furin site mutant S-Trimer fr... -

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Basic information

Entry
Database: PDB / ID: 7e7b
TitleCryo-EM structure of the SARS-CoV-2 furin site mutant S-Trimer from a subunit vaccine candidate
ComponentsSpike glycoprotein,Collagen alpha-1(I) chain
KeywordsVIRAL PROTEIN / spike protein / COVID-19 / vaccine
Function / homology
Function and homology information


cellular response to fluoride / collagen type I trimer / cellular response to vitamin E / tooth mineralization / Anchoring fibril formation / Crosslinking of collagen fibrils / collagen biosynthetic process / Collagen chain trimerization / Defective VWF binding to collagen type I / platelet-derived growth factor binding ...cellular response to fluoride / collagen type I trimer / cellular response to vitamin E / tooth mineralization / Anchoring fibril formation / Crosslinking of collagen fibrils / collagen biosynthetic process / Collagen chain trimerization / Defective VWF binding to collagen type I / platelet-derived growth factor binding / bone trabecula formation / Enhanced cleavage of VWF variant by ADAMTS13 / Defective VWF cleavage by ADAMTS13 variant / Enhanced binding of GP1BA variant to VWF multimer:collagen / Defective binding of VWF variant to GPIb:IX:V / extracellular matrix structural constituent conferring tensile strength / intramembranous ossification / Extracellular matrix organization / embryonic skeletal system development / cartilage development involved in endochondral bone morphogenesis / Collagen biosynthesis and modifying enzymes / skin morphogenesis / collagen-activated tyrosine kinase receptor signaling pathway / endochondral ossification / Platelet Adhesion to exposed collagen / cellular response to fibroblast growth factor stimulus / collagen fibril organization / negative regulation of cell-substrate adhesion / response to steroid hormone / face morphogenesis / Scavenging by Class A Receptors / Assembly of collagen fibrils and other multimeric structures / MET activates PTK2 signaling / Syndecan interactions / GP1b-IX-V activation signalling / skin development / blood vessel development / RUNX2 regulates osteoblast differentiation / Platelet Aggregation (Plug Formation) / Collagen degradation / Non-integrin membrane-ECM interactions / protein localization to nucleus / ECM proteoglycans / response to hyperoxia / Integrin cell surface interactions / response to mechanical stimulus / positive regulation of epithelial to mesenchymal transition / cellular response to retinoic acid / cellular response to transforming growth factor beta stimulus / GPVI-mediated activation cascade / response to cAMP / cellular response to epidermal growth factor stimulus / visual perception / extracellular matrix organization / ossification / secretory granule / skeletal system development / cellular response to amino acid stimulus / Cell surface interactions at the vascular wall / cellular response to glucose stimulus / sensory perception of sound / response to insulin / response to hydrogen peroxide / cellular response to mechanical stimulus / osteoblast differentiation / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / positive regulation of canonical Wnt signaling pathway / protein transport / cellular response to tumor necrosis factor / response to estradiol / protease binding / collagen-containing extracellular matrix / 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 / positive regulation of cell migration / response to xenobiotic stimulus / endoplasmic reticulum lumen / 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
Similarity search - Function
Fibrillar collagen, C-terminal / Fibrillar collagen C-terminal domain / Fibrillar collagen C-terminal non-collagenous (NC1) domain profile. / Fibrillar collagens C-terminal domain / : / von Willebrand factor type C domain / VWFC domain signature. / VWFC domain profile. / von Willebrand factor (vWF) type C domain / VWFC domain ...Fibrillar collagen, C-terminal / Fibrillar collagen C-terminal domain / Fibrillar collagen C-terminal non-collagenous (NC1) domain profile. / Fibrillar collagens C-terminal domain / : / von Willebrand factor type C domain / VWFC domain signature. / VWFC domain profile. / von Willebrand factor (vWF) type C domain / VWFC domain / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / 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
9-OCTADECENOIC ACID / Chem-VCG / Collagen alpha-1(I) chain / Spike glycoprotein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsZheng, S. / Ma, J.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: J Virol / Year: 2021
Title: Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19.
Authors: Jiahao Ma / Danmei Su / Yinyan Sun / Xueqin Huang / Ying Liang / Linqiang Fang / Yan Ma / Wenhui Li / Peng Liang / Sanduo Zheng /
Abstract: Within a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people worldwide with a death toll over 2 million. Vaccination ...Within a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people worldwide with a death toll over 2 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Å and 2.6 Å respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. The tightly closed conformation is stabilized by fatty acid and polysorbate 80 binding at the receptor binding domains (RBDs) and the N terminal domains (NTDs) respectively. Additionally, we identified an important pH switch in the WT S-Trimer that shows dramatic conformational change and accounts for its increased stability at lower pH. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.Effective vaccine against SARS-CoV-2 is critical to end the COVID-19 pandemic. Here, using Trimer-Tag technology, we are able to produce stable and large quantities of WT S-Trimer, a subunit vaccine candidate for COVID-19 with high safety and efficacy from animal and Phase 1 clinical trial studies. Cryo-EM structures of the S-Trimer subunit vaccine candidate show that it predominately adopts tightly closed pre-fusion state, and resembles that of the native and full-length spike in detergent, confirming its structural integrity. WT S-Trimer is currently being evaluated in global Phase 2/3 clinical trial. Combining with published structures of the S protein, we also propose a model to dissect the conformation change of the spike protein before receptor binding.
History
DepositionFeb 25, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 24, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_initial_refinement_model
Item: _citation.journal_volume / _citation_author.identifier_ORCID ..._citation.journal_volume / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Spike glycoprotein,Collagen alpha-1(I) chain
B: Spike glycoprotein,Collagen alpha-1(I) chain
C: Spike glycoprotein,Collagen alpha-1(I) chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)526,76663
Polymers504,2353
Non-polymers22,53260
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Spike glycoprotein,Collagen alpha-1(I) chain / S glycoprotein / E2 / Peplomer protein / Alpha-1 type I collagen


Mass: 168078.203 Da / Num. of mol.: 3 / Mutation: R685A
Source method: isolated from a genetically manipulated source
Details: Chimeric protein
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2, (gene. exp.) Homo sapiens (human)
Gene: S, 2, COL1A1 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P0DTC2, UniProt: P02452

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Sugars , 2 types, 54 molecules

#2: 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.: 39
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
#3: 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.: 15 / 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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Non-polymers , 3 types, 42 molecules

#4: Chemical ChemComp-ELA / 9-OCTADECENOIC ACID


Mass: 282.461 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C18H34O2
#5: Chemical ChemComp-VCG / 2-hydroxyethyl 2-deoxy-3,5-bis-O-(2-hydroxyethyl)-6-O-(2-{[(9E)-octadec-9-enoyl]oxy}ethyl)-alpha-L-xylo-hexofuranoside / Polysorbate 80


Mass: 604.813 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C32H60O10
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: SARS-CoV-2 spike protein fused to the C-terminal region of human type 1a collagen
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Severe acute respiratory syndrome coronavirus 22697049
21Homo sapiens (human)9606
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 282 K
Details: blot time 2 seconds, blot force 4, waiting time 8 seconds

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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 FIELD / Nominal magnification: 64000 X / Calibrated defocus min: 600 nm / Calibrated defocus max: 2800 nm / Cs: 0 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2000

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4CTFFIND4CTF correctionCTF correction
7Coot0.9-premodel fittingmodel built
11RELION3.0.7classificationclassification
12RELION3.0.73D reconstructionreconstruction
13PHENIX1.16-3549model refinementmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1504770
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 384013 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6VXX

6vxx


Accession code: 6VXX / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00927951
ELECTRON MICROSCOPYf_angle_d0.66237983
ELECTRON MICROSCOPYf_dihedral_angle_d10.31716320
ELECTRON MICROSCOPYf_chiral_restr0.0484521
ELECTRON MICROSCOPYf_plane_restr0.0044827

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