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- PDB-6x6p: Characterization of the SARS-CoV-2 S Protein: Biophysical, Bioche... -

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Entry
Database: PDB / ID: 6x6p
TitleCharacterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
ComponentsSpike glycoprotein
KeywordsVIRAL PROTEIN / coronavirus / SARS-CoV-2 / SARS-CoV / spike glycoprotein / fusion protein / trimer
Function / homology
Function and homology information


symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / viral translation / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion ...symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / viral translation / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / membrane fusion / Attachment and Entry / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont entry into host cell / 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 glycoprotein, N-terminal domain / 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, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. ...Spike glycoprotein, N-terminal domain / 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, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / 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 / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2 superfamily, coronavirus / 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, coronavirus / Coronavirus spike glycoprotein S2 / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsHerrera, N.G. / Morano, N.C. / Celikgil, A. / Georgiev, G.I. / Malonis, R. / Lee, J.H. / Tong, K. / Vergnolle, O. / Massimi, A. / Yen, L.Y. ...Herrera, N.G. / Morano, N.C. / Celikgil, A. / Georgiev, G.I. / Malonis, R. / Lee, J.H. / Tong, K. / Vergnolle, O. / Massimi, A. / Yen, L.Y. / Noble, A.J. / Kopylov, M. / Bonanno, J.B. / Garrett-Thompson, S.C. / Hayes, D.B. / Brenowitz, M. / Garforth, S.J. / Eng, E.T. / Lai, J.R. / Almo, S.C.
CitationJournal: bioRxiv / Year: 2020
Title: Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.
Authors: Natalia G Herrera / Nicholas C Morano / Alev Celikgil / George I Georgiev / Ryan J Malonis / James H Lee / Karen Tong / Olivia Vergnolle / Aldo B Massimi / Laura Y Yen / Alex J Noble / ...Authors: Natalia G Herrera / Nicholas C Morano / Alev Celikgil / George I Georgiev / Ryan J Malonis / James H Lee / Karen Tong / Olivia Vergnolle / Aldo B Massimi / Laura Y Yen / Alex J Noble / Mykhailo Kopylov / Jeffrey B Bonanno / Sarah C Garrett-Thomson / David B Hayes / Robert H Bortz / Ariel S Wirchnianski / Catalina Florez / Ethan Laudermilch / Denise Haslwanter / J Maximilian Fels / M Eugenia Dieterle / Rohit K Jangra / Jason Barnhill / Amanda Mengotto / Duncan Kimmel / Johanna P Daily / Liise-Anne Pirofski / Kartik Chandran / Michael Brenowitz / Scott J Garforth / Edward T Eng / Jonathan R Lai / Steven C Almo /
Abstract: Coronavirus disease 2019 ( ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( ), and there is a critical need to produce large quantities of high- ...Coronavirus disease 2019 ( ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
History
DepositionMay 28, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_entity_id / _chem_comp.name / _pdbx_entity_nonpoly.entity_id / _pdbx_entity_nonpoly.name / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Jan 27, 2021Group: Refinement description / Structure summary
Category: chem_comp / entity ...chem_comp / entity / entity_name_com / struct_ncs_dom_lim
Item: _chem_comp.pdbx_synonyms / _entity.pdbx_description / _entity_name_com.name
Revision 2.2Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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
B: Spike glycoprotein
C: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)434,86648
Polymers422,4733
Non-polymers12,39345
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain "B"
21chain "C"
31chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
111ALASERB1 - 1017
211ALASERC1 - 1017
311ALASERA1 - 1017

NCS oper:
IDCodeMatrixVector
1given(-0.500074653982, -0.865982291205, -0.000108465061909), (0.865982294605, -0.500074659187, 2.5875439059E-5), (-7.66483008702E-5, -8.09891719627E-5, 0.999999993783)480.655587524, 128.800597837, 0.0318858294733
2given(-0.500926006666, 0.865490043122, -0.000347996512365), (-0.865490094047, -0.500926039489, -8.32962411061E-6), (-0.000181529721426, 0.000297015008872, 0.999999939415)129.149667515, 480.706760068, -0.0561034599074

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Components

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein / SARS-CoV-2 spike glycoprotein


Mass: 140824.406 Da / Num. of mol.: 3 / Mutation: R682G R683S R685S K986P V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P0DTC2
#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.: 12
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: 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.: 33
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
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: Severe acute respiratory syndrome coronavirus 2 / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Details of virusEmpty: YES / Enveloped: YES / Isolate: OTHER / Type: VIRION
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1250 mMsodium chlorideNaCl1
250 mMTris1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 %

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 66.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1131

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
EM software
IDNameVersionCategory
1cryoSPARC2.14.2particle selection
2RELIONimage acquisition
4cryoSPARC2.14.2CTF correction
7Coot0.8.9.2model fitting
9PHENIX1.18-3845model refinement
10cryoSPARC2.14.2initial Euler assignment
11cryoSPARC2.14.2final Euler assignment
12cryoSPARC2.14.2classification
13cryoSPARC2.14.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 75582
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54395 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 120 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC
Atomic model buildingPDB-ID: 6VXX

6vxx


Pdb chain-ID: A / Accession code: 6VXX / Pdb chain residue range: 27-1147 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 120.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.011925152
ELECTRON MICROSCOPYf_angle_d1.018434233
ELECTRON MICROSCOPYf_chiral_restr0.06334074
ELECTRON MICROSCOPYf_plane_restr0.0064350
ELECTRON MICROSCOPYf_dihedral_angle_d13.38333600
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
12BELECTRON MICROSCOPYNCS constraints0.000705603610554
13BELECTRON MICROSCOPYNCS constraints0.0347396776632

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