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- EMDB-20671: PEDV spike dissociated S1 ring -

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Basic information

Entry
Database: EMDB / ID: EMD-20671
TitlePEDV spike dissociated S1 ring
Map dataPEDV dissociated S1 ring
Sample
  • Complex: Homotrimeric PEDV S1 ring that has dissociated from S2
    • Protein or peptide: PEDV S1
Function / homology
Function and homology information


endocytosis involved in viral entry into host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion membrane / membrane
Similarity search - Function
Spike glycoprotein, Alphacoronavirus / Spike glycoprotein S1, coronavirus / Coronavirus spike glycoprotein S1 / Spike glycoprotein S2, coronavirus, C-terminal / Coronavirus spike glycoprotein S2, intravirion / 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, Alphacoronavirus / Spike glycoprotein S1, coronavirus / Coronavirus spike glycoprotein S1 / Spike glycoprotein S2, coronavirus, C-terminal / Coronavirus spike glycoprotein S2, intravirion / 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
Biological speciesPorcine epidemic diarrhea virus CV777
Methodsingle particle reconstruction / cryo EM / Resolution: 9.4 Å
AuthorsWrapp D / McLellan JS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01-AI127521 United States
CitationJournal: J Virol / Year: 2019
Title: The 3.1-Angstrom Cryo-electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation.
Authors: Daniel Wrapp / Jason S McLellan /
Abstract: Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that has a significant agricultural and economic impact due to the high mortality rate associated with infection of neonatal piglets. ...Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that has a significant agricultural and economic impact due to the high mortality rate associated with infection of neonatal piglets. Like other coronaviruses, PEDV makes use of a large, trimeric spike (S) glycoprotein to mediate membrane fusion and gain entry into host cells. Despite the importance of the spike protein in viral entry and host immune responses, high-resolution structural information concerning this large macromolecular machine has been difficult to obtain. Here, we report the cryo-electron microscopy structure of the PEDV S protein in the prefusion conformation at a resolution of 3.1 Å. Our studies revealed that the sialic acid-binding domain at the N terminus of the S1 subunit has an orientation that is substantially different from that observed in the previously determined spike structure from human alphacoronavirus NL63. We also observed dissociated S1 subunit trimers wherein the putative receptor-binding domains exist in a conformation differing from that observed in the intact spike proteins, suggesting that the PEDV receptor-binding domain undergoes conformational rearrangements akin to those that have been described in the related betacoronaviruses. Collectively, these data provide new insights into the biological processes that mediate alphacoronavirus attachment, receptor engagement, and fusion triggering while also identifying a source of conformational heterogeneity that could be manipulated to improve PEDV vaccine antigens. Coronavirus spike proteins are large, densely glycosylated macromolecular machines that mediate receptor binding and membrane fusion to facilitate entry into host cells. This report describes the atomic-resolution structure of the spike protein from porcine epidemic diarrhea virus, a pathogenic alphacoronavirus that causes severe agricultural damage. The structure reveals a novel position for the sialic acid-binding attachment domain in the intact spike. We also observed shed fusion-suppressive capping subunits that displayed the putative receptor-binding domain in an accessible conformation. These observations provide a basis for understanding the molecular mechanisms that drive the earliest stages of alphacoronavirus infection and will inform future efforts to rationally design vaccines.
History
DepositionSep 3, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 18, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.105
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.105
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20671.png

Downloads & links

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Map

FileDownload / File: emd_20671.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPEDV dissociated S1 ring
Voxel sizeX=Y=Z: 1.075 Å
Density
Contour LevelBy AUTHOR: 0.105 / Movie #1: 0.105
Minimum - Maximum-0.071216404 - 0.35056978
Average (Standard dev.)-0.00030948324 (±0.011902764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 464.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0751.0751.075
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z464.400464.400464.400
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS432432432
D min/max/mean-0.0710.351-0.000

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Supplemental data

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Half map: PEDV dissociated S1 ring half map B

Fileemd_20671_half_map_1.map
AnnotationPEDV dissociated S1 ring half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: PEDV dissociated S1 ring half map A

Fileemd_20671_half_map_2.map
AnnotationPEDV dissociated S1 ring half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homotrimeric PEDV S1 ring that has dissociated from S2

EntireName: Homotrimeric PEDV S1 ring that has dissociated from S2
Components
  • Complex: Homotrimeric PEDV S1 ring that has dissociated from S2
    • Protein or peptide: PEDV S1

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Supramolecule #1: Homotrimeric PEDV S1 ring that has dissociated from S2

SupramoleculeName: Homotrimeric PEDV S1 ring that has dissociated from S2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Porcine epidemic diarrhea virus CV777
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: PEDV S1

MacromoleculeName: PEDV S1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus CV777
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRSLIYFWLL LPVLPTLSLP QDVTRCQSTT NFRRFFSKFN VQAPAVVVLG GYLPSMNSSS WYCGTGIETA SGVHGIFLSY IDSGQGFEIG ISQEPFDPSG YQLYLHKATN GNTNAIARLR ICQFPDNKTL GPTVNDVTTG RNCLFNKAIP AYMRDGKDIV VGITWDNDRV ...String:
MRSLIYFWLL LPVLPTLSLP QDVTRCQSTT NFRRFFSKFN VQAPAVVVLG GYLPSMNSSS WYCGTGIETA SGVHGIFLSY IDSGQGFEIG ISQEPFDPSG YQLYLHKATN GNTNAIARLR ICQFPDNKTL GPTVNDVTTG RNCLFNKAIP AYMRDGKDIV VGITWDNDRV TVFADKIYHF YLKNDWSRVA TRCYNRRSCA MQYVYTPTYY MLNVTSAGED GIYYEPCTAN CTGYAANVFA TDSNGHIPEG FSFNNWFLLS NDSTLLHGKV VSNQPLLVNC LLAIPKIYGL GQFFSFNHTM DGVCNGAAVD RAPEALRFNI NDTSVILAEG SIVLHTALGT NLSFVCSNSS DPHLAIFAIP LGATEVPYYC FLKVDTYNST VYKFLAVLPP TVREIVITKY GDVYVNGFGY LHLGLLDAVT INFTGHGTDD DVSGFWTIAS TNFVDALIEV QGTSIQRILY CDDPVSQLKC SQVAFDLDDG FYPISSRNLL SHEQPISFVT LPSFNDHSFV NITVSAAFGG LSSANLVASD TTINGFSSFC VDTRQFTITL FYNVTNSYGY VSKSQDSNCP FTLQSVNDYL SFSKFCVSTS LLAGACTIDL FGYPAFGSGV KLTSLYFQFT KGELITGTPK PLEGITDVSF MTLDVCTKYT IYGFKGEGII TLTNSSILAG VYYTSDSGQL LAFKNVTSGA VYSVTPCSFS EQAAYVNDDI VGVISSLSNS TFNNTRELPG FFYHSNDGSN CTEPVLVYSN IGVCKSGSIG YVPSQYGQVK IAPTVTGNIS IPTNFSMSIR TEYLQLYNTP VSVDCATYVC NGNSRCKQLL TQYTAACKTI ESALQLSARL ESVEVNSMLT ISEEALQLAT ISSFNGDGYN FTNVLGASVY DPASGRVVQK RSVIEDLLFN KVVTNGLGTV DEDYKRCSNG RSVADLVCAQ YYSGVMVLPG VVDAEKLHMY SASLIGGMAL GGITAAAALP FSYAVQARLN YLALQTDVLQ RNQQLLAESF NSAIGNITSA FESVKEAISQ TSKGLNTVAH ALTKVQEVVN SQGSALNQLT VQLQHNFQAI SSSIDDIYSR LDILSADVQV DRLITGRLSA LNAFVAQTLT KYTEVQASRK LAQQKVNECV KSQSQRYGFC GGDGEHIFSL VQAAPQGLLF LHTVLVPGDF VNVLAIAGLC VNGEIALTLR EPGLVLFTHE LQTYTATEYF VSSRRMFEPR KPTVSDFVQI ESCVVTYVNL TSDQLPDVIP DYIDVNKTLD EILASLPNRT GPSLPLDVFN ATYLNLTGEI ADLEQRSESL RNTTEELRSL INNINNTLVD LEWLNRVETG SGYIPEAPRD GQAYVRKDGE WVLLSTFLGR SLEVLFQGPG HHHHHHHHSA WSHPQFEKGG GSGGGGSGGS AWSHPQFEK

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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

Concentration0.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
2.0 mMNH2C(CH2OH)3-HClTris-HCl
200.0 mMNaClSodium chloride
0.02 %NaN3Sodium azide
0.01 %(C6.2H10.3O1.35N0.65Na0.35)-72Amphipol A8-35
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 6 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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

CTF correctionSoftware - Name: Warp
Startup modelType of model: OTHER / Details: Ab initio model generated using cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.4.6) / Number images used: 28993
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.4.6)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.4.6)
Final 3D classificationNumber classes: 50 / Software - Name: cryoSPARC (ver. 2.4.6)
FSC plot (resolution estimation)

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