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- EMDB-33462: Structure of SARS-CoV-2 Spike Protein with Engineered x3 Disulfid... -

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Basic information

Entry
Database: EMDB / ID: EMD-33462
TitleStructure of SARS-CoV-2 Spike Protein with Engineered x3 Disulfide (x3(D427C, V987C) and single Arg S1/S2 cleavage site), incubated in Low pH after 40-Day Storage in PBS, Closed Conformation
Map data
Sample
  • Complex: Severe acute respiratory syndrome coronavirus 2 Spike protein
    • Protein or peptide: Spike glycoprotein
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsQu K / Chen Q / Ciazynska KA / Liu B / Zhang X / Wang J / He Y / Guan J / He J / Liu T ...Qu K / Chen Q / Ciazynska KA / Liu B / Zhang X / Wang J / He Y / Guan J / He J / Liu T / Carter AP / Xiong X / Briggs JAG
Funding supportEuropean Union, United Kingdom, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-CoG-648432European Union
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1011 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
CitationJournal: PLoS Pathog / Year: 2022
Title: Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike.
Authors: Kun Qu / Qiuluan Chen / Katarzyna A Ciazynska / Banghui Liu / Xixi Zhang / Jingjing Wang / Yujie He / Jiali Guan / Jun He / Tian Liu / Xiaofei Zhang / Andrew P Carter / Xiaoli Xiong / John A G Briggs /
Abstract: The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. ...The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.
History
DepositionMay 18, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateSep 7, 2022-
Current statusSep 7, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33462.png

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Map

FileDownload / File: emd_33462.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 360 pix.
= 381.96 Å		1.06 Å/pix.
x 360 pix.
= 381.96 Å		1.06 Å/pix.
x 360 pix.
= 381.96 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.061 Å
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Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.091567725 - 0.17834206
Average (Standard dev.)2.9459003e-05 (±0.0028684242)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_33462_half_map_1.map
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Half map: #2

Fileemd_33462_half_map_2.map
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Sample components

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Entire : Severe acute respiratory syndrome coronavirus 2 Spike protein

EntireName: Severe acute respiratory syndrome coronavirus 2 Spike protein
Components
  • Complex: Severe acute respiratory syndrome coronavirus 2 Spike protein
    • Protein or peptide: Spike glycoprotein

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Supramolecule #1: Severe acute respiratory syndrome coronavirus 2 Spike protein

SupramoleculeName: Severe acute respiratory syndrome coronavirus 2 Spike protein
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ETGTQCVNLT TRTQLPPAYT NSFTRGVYYP DKVFRSSVLH STQDLFLPFF SNVTWFHAIH VSGTNGTKRF DNPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV YSSANNCTFE YVSQPFLMDL ...String:
ETGTQCVNLT TRTQLPPAYT NSFTRGVYYP DKVFRSSVLH STQDLFLPFF SNVTWFHAIH VSGTNGTKRF DNPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV YSSANNCTFE YVSQPFLMDL EGKQGNFKNL REFVFKNIDG YFKIYSKHTP INLVRDLPQG FSALEPLVDL PIGINITRFQ TLLALHRSYL TPGDSSSGWT AGAAAYYVGY LQPRTFLLKY NENGTITDAV DCALDPLSET KCTLKSFTVE KGIYQTSNFR VQPTESIVRF PNITNLCPFG EVFNATRFAS VYAWNRKRIS NCVADYSVLY NSASFSTFKC YGVSPTKLND LCFTNVYADS FVIRGDEVRQ IAPGQTGKIA DYNYKLPCDF TGCVIAWNSN NLDSKVGGNY NYLYRLFRKS NLKPFERDIS TEIYQAGSTP CNGVEGFNCY FPLQSYGFQP TNGVGYQPYR VVVLSFELLH APATVCGPKK STNLVKNKCV NFNFNGLTGT GVLTESNKKF LPFQQFGRDI ADTTDAVRDP QTLEILDITP CSFGGVSVIT PGTNTSNQVA VLYQDVNCTE VPVAIHADQL TPTWRVYSTG SNVFQTRAGC LIGAEHVNNS YECDIPIGAG ICASYQTQTN SRSVASQSII AYTMSLGAEN SVAYSNNSIA IPTNFTISVT TEILPVSMTK TSVDCTMYIC GDSTECSNLL LQYGSFCTQL NRALTGIAVE QDKNTQEVFA QVKQIYKTPP IKDFGGFNFS QILPDPSKPS KRSFIEDLLF NKVTLADAGF IKQYGDCLGD IAARDLICAQ KFNGLTVLPP LLTDEMIAQY TSALLAGTIT SGWTFGAGAA LQIPFAMQMA YRFNGIGVTQ NVLYENQKLI ANQFNSAIGK IQDSLSSTAS ALGKLQDVVN QNAQALNTLV KQLSSNFGAI SSVLNDILSR LDKCEAEVQI DRLITGRLQS LQTYVTQQLI RAAEIRASAN LAATKMSECV LGQSKRVDFC GKGYHLMSFP QSAPHGVVFL HVTYVPAQEK NFTTAPAICH DGKAHFPREG VFVSNGTHWF VTQRNFYEPQ IITTDNTFVS GNCDVVIGIV NNTVYDPLQP ELDSFKEELD KYFKNHTSPD VDLGDISGIN ASVVNIQKEI DRLNEVAKNL NESLIDLQEL GKYEQYIKGS GRENLYFQGG GGSGYIPEAP RDGQAYVRKD GEWVLLSTFL GHHHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 5
GridModel: C-flat-2/2 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 1698 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1505895 / Details: Template picking
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 42621
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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