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- PDB-7a4n: Cryo-EM structure of a prefusion stabilized SARS-CoV-2 Spike (D61... -

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Basic information

Entry
Database: PDB / ID: 7a4n
TitleCryo-EM structure of a prefusion stabilized SARS-CoV-2 Spike (D614N, R682S, R685G, A892P, A942P and V987P)(S-closed trimer)
ComponentsSpike glycoprotein,Fibritin
KeywordsVIRAL PROTEIN / SARS-CoV-2 / virology / COVID-19 / class I fusion proteins / S glycoprotein / cryo-EM / prefusion / corona
Function / homology
Function and homology information


virion component / 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 ...virion component / 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 / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane
Similarity search - Function
Fibritin C-terminal / Fibritin C-terminal region / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus ...Fibritin C-terminal / Fibritin C-terminal region / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, 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, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Spike glycoprotein / Fibritin
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Enterobacteria phage T4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsRutten, L. / Renault, L.L.R. / Juraszek, J. / Langedijk, J.P.M.
CitationJournal: Nat Commun / Year: 2021
Title: Stabilizing the closed SARS-CoV-2 spike trimer.
Authors: Jarek Juraszek / Lucy Rutten / Sven Blokland / Pascale Bouchier / Richard Voorzaat / Tina Ritschel / Mark J G Bakkers / Ludovic L R Renault / Johannes P M Langedijk /
Abstract: The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to ...The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.
History
DepositionAug 20, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Spike glycoprotein,Fibritin
B: Spike glycoprotein,Fibritin
C: Spike glycoprotein,Fibritin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)432,23321
Polymers427,6423
Non-polymers4,59118
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 27 through 339 or resid 341...
d_2ens_1(chain "A" and (resid 27 through 339 or resid 341...
d_3ens_1(chain "C" and (resid 27 through 339 or resid 341...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ALAGLYB1 - 214
d_12ens_1VALALAB218 - 221
d_13ens_1ARGGLYB225 - 260
d_14ens_1SERPHEB264 - 310
d_15ens_1GLYASPB314 - 325
d_16ens_1LYSILEB329 - 353
d_17ens_1THRSERB357 - 975
d_18ens_1NAGNAGK
d_19ens_1NAGNAGL
d_110ens_1NAGNAGM
d_111ens_1NAGNAGN
d_112ens_1NAGNAGO
d_113ens_1NAGNAGP
d_114ens_1NAGNAGQ
d_21ens_1ALAGLYA1 - 214
d_22ens_1VALALAA218 - 221
d_23ens_1ARGGLYA225 - 260
d_24ens_1SERPHEA264 - 310
d_25ens_1GLYASPA314 - 325
d_26ens_1LYSILEA329 - 353
d_27ens_1THRSERA357 - 975
d_28ens_1NAGNAGD
d_29ens_1NAGNAGE
d_210ens_1NAGNAGF
d_211ens_1NAGNAGG
d_212ens_1NAGNAGH
d_213ens_1NAGNAGI
d_214ens_1NAGNAGJ
d_31ens_1ALAGLYC1 - 214
d_32ens_1VALALAC218 - 221
d_33ens_1ARGGLYC225 - 260
d_34ens_1SERPHEC264 - 310
d_35ens_1GLYASPC314 - 325
d_36ens_1LYSILEC329 - 353
d_37ens_1THRSERC357 - 975
d_38ens_1NAGNAGR
d_39ens_1NAGNAGS
d_310ens_1NAGNAGT
d_311ens_1NAGNAGU

NCS oper:
IDCodeMatrixVector
1given(0.736198759864, 0.652130396929, 0.180934605245), (0.114225036659, -0.383249691051, 0.916554589378), (0.667056239739, -0.654099090131, -0.356637565766)-29.4450027517, 23.3702203099, 64.7544727057
2given(0.735548820151, 0.113901538383, 0.667828101182), (0.652845587257, -0.38253564321, -0.653803579734), (0.180998818708, 0.916893080587, -0.355733757744)-24.1450837631, 70.4007156913, 6.91715272665

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Components

#1: Protein Spike glycoprotein,Fibritin / S glycoprotein / E2 / Peplomer protein / Collar protein / Whisker antigen control protein


Mass: 142547.344 Da / Num. of mol.: 3 / Mutation: D614N,R682S,R685G,A892P,A942P,V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2, (gene. exp.) Enterobacteria phage T4 (virus)
Gene: S, 2, wac / Plasmid: pCDNA2004 / Cell line (production host): Expi293F / Organ (production host): kidney / Production host: Homo sapiens (human) / References: UniProt: P0DTC2, UniProt: P10104
#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.: 3
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 / N-Acetylglucosamine


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
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: S closed protein trimer / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Enterobacteria phage T4 (virus)10665
31Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)Organism: Homo sapiens (human) / Cell: kidney / Plasmid: pCDNA2004
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtrisaminomethaneTris1
2150 mMSodium ChlorideNaCLSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Calibrated defocus min: 400 nm / Calibrated defocus max: 2700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9760
Details: Movies with 50 frames were acquired in super resolution counting mode.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2.6image acquisition
4CTFFIND4.1.18CTF correction
7Coot0.9model fitting
10RELION3.1 betafinal Euler assignment
11RELION3.1 betaclassification
12RELION3.1 beta3D reconstruction
13PHENIX1.18.261model refinement
Image processingDetails: collected movies were subjected to beam induced drift correction using MotionCor2 and downsampled at this stage
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1391000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 315000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00823469
ELECTRON MICROSCOPYf_angle_d0.67131983
ELECTRON MICROSCOPYf_dihedral_angle_d9.9353273
ELECTRON MICROSCOPYf_chiral_restr0.0493714
ELECTRON MICROSCOPYf_plane_restr0.0054134
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints0.000705974410502
ens_1d_3BELECTRON MICROSCOPYNCS constraints0.000708097021524

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