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- PDB-6zow: SARS-CoV-2 spike in prefusion state -

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Basic information

Entry
Database: PDB / ID: 6zow
TitleSARS-CoV-2 spike in prefusion state
ComponentsSpike glycoprotein
KeywordsVIRAL PROTEIN / Spike / Prefusion
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / host cell surface receptor binding / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane
Spike glycoprotein S2, coronavirus / Spike receptor binding domain, betacoronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike receptor binding domain superfamily, coronavirus
Spike glycoprotein
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMartinez, M. / Marabini, R. / Carazo, J.M.
Funding support Spain, European Union, United States, 5items
OrganizationGrant numberCountry
Spanish National Research CouncilPIE/COVID-19 202020E079 Spain
Spanish Ministry of Science, Innovation, and UniversitiesSEV 2017-0712 Spain
European Research Council (ERC)ERC - 2018 - SyG, Proposal: 810057European Union
National Institutes of Health/National Library of Medicine (NIH/NLM)GM125769 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)R01-AI127521 United States
CitationJournal: bioRxiv / Year: 2020
Title: Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures.
Abstract: With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We ...With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spike glycoprotein
C: Spike glycoprotein
B: Spike glycoprotein
a: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)583,52154
Polymers565,0534
Non-polymers18,46750
Water724
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area45670 Å2
ΔGint76 kcal/mol
Surface area137580 Å2
MethodPISA

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Components

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Protein , 1 types, 4 molecules ACBa

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


Mass: 141263.344 Da / Num. of mol.: 4 / Mutation: K986P, E987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Plasmid: palphaH / Cell line (production host): FreeStyle293F / Production host: Homo sapiens (human) / References: UniProt: P0DTC2

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Sugars , 6 types, 47 molecules

#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


GlcNAc

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GlcNAc

Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 16
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
#3: Polysaccharide
alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


GlcNAc

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GlcNAc

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Man

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Man

Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


GlcNAc

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GlcNAc

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Man

Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 6
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-(1-6)-2-acetamido-2-deoxy-beta-D-glucopyranose


GlcNAc

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GlcNAc

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Man

Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-6DGlcpNAcb1-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/a6-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(6+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#6: 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.: 18
Source method: isolated from a genetically manipulated source
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
#7: Sugar ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose / Mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 7 molecules

#8: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: Spike homotrimer in prefusion state / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human) / Cell: FreeStyle293F / Plasmid: palphaH
Buffer solutionpH: 8
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 36 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
7UCSF Chimeramodel fittingRigid fitting
8PHENIXmodel fittingRigid fitting
9CCP4 packagemodel fittingFlexible fitting
11PHENIX1.17model refinement
14RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 203000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial models 6VSB and 6VYB were used for flexible fitting, whereas 6YLA was used for rigid fitting of the RBD domain of chain A, that we have called a in the final structure.
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
16VSBA11-335
26VSBA1526-1208
36VSBB11-1208
46VSBC11-1208
56VYBA11-335
66VYBA1526-1208
76VYBB11-1208
86VYBC11-1208
96YLAA1336-525

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