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- EMDB-20672: Prefusion structure of PEDV spike -

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Basic information

Entry
Database: EMDB / ID: EMD-20672
TitlePrefusion structure of PEDV spike
Map dataPEDV S sharpened map using LocalDeblur
Sample
  • Complex: Homotrimeric complex of PEDV spike
    • Protein or peptide: Spike glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / Porcine epidemic diarrhea virus (strain CV777)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsWrapp D / McLellan JS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01-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: 0.43
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.43
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6u7k
  • Surface level: 0.43
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20672.png

Downloads & links

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Map

FileDownload / File: emd_20672.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPEDV S sharpened map using LocalDeblur
Voxel sizeX=Y=Z: 1.075 Å
Density
Contour LevelBy AUTHOR: 0.43 / Movie #1: 0.43
Minimum - Maximum-0.03089493 - 4.2172313
Average (Standard dev.)0.0005822824 (±0.04567407)
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/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS432432432
D min/max/mean-0.0314.2170.001

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Supplemental data

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Additional map: PEDV S unsharpened map

Fileemd_20672_additional.map
AnnotationPEDV S unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: PEDV S half map A

Fileemd_20672_half_map_1.map
AnnotationPEDV S half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: PEDV S half map B

Fileemd_20672_half_map_2.map
AnnotationPEDV S half map B
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 complex of PEDV spike

EntireName: Homotrimeric complex of PEDV spike
Components
  • Complex: Homotrimeric complex of PEDV spike
    • Protein or peptide: Spike glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Homotrimeric complex of PEDV spike

SupramoleculeName: Homotrimeric complex of PEDV spike / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Porcine epidemic diarrhea virus CV777
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 452 KDa

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus (strain CV777) / Strain: CV777
Molecular weightTheoretical: 152.906906 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRSLIYFWLL LPVLPTLSLP QDVTRCQSTT NFRRFFSKFN VQAPAVVVLG GYLPSMNSSS WYCGTGIETA SGVHGIFLSY IDSGQGFEI GISQEPFDPS GYQLYLHKAT NGNTNAIARL RICQFPDNKT LGPTVNDVTT GRNCLFNKAI PAYMRDGKDI V VGITWDND ...String:
MRSLIYFWLL LPVLPTLSLP QDVTRCQSTT NFRRFFSKFN VQAPAVVVLG GYLPSMNSSS WYCGTGIETA SGVHGIFLSY IDSGQGFEI GISQEPFDPS GYQLYLHKAT NGNTNAIARL RICQFPDNKT LGPTVNDVTT GRNCLFNKAI PAYMRDGKDI V VGITWDND RVTVFADKIY HFYLKNDWSR VATRCYNRRS CAMQYVYTPT YYMLNVTSAG EDGIYYEPCT ANCTGYAANV FA TDSNGHI PEGFSFNNWF LLSNDSTLLH GKVVSNQPLL VNCLLAIPKI YGLGQFFSFN HTMDGVCNGA AVDRAPEALR FNI NDTSVI LAEGSIVLHT ALGTNLSFVC SNSSDPHLAI FAIPLGATEV PYYCFLKVDT YNSTVYKFLA VLPPTVREIV ITKY GDVYV NGFGYLHLGL LDAVTINFTG HGTDDDVSGF WTIASTNFVD ALIEVQGTSI QRILYCDDPV SQLKCSQVAF DLDDG FYPI SSRNLLSHEQ PISFVTLPSF NDHSFVNITV SAAFGGLSSA NLVASDTTIN GFSSFCVDTR QFTITLFYNV TNSYGY VSK SQDSNCPFTL QSVNDYLSFS KFCVSTSLLA GACTIDLFGY PAFGSGVKLT SLYFQFTKGE LITGTPKPLE GITDVSF MT LDVCTKYTIY GFKGEGIITL TNSSILAGVY YTSDSGQLLA FKNVTSGAVY SVTPCSFSEQ AAYVNDDIVG VISSLSNS T FNNTRELPGF FYHSNDGSNC TEPVLVYSNI GVCKSGSIGY VPSQYGQVKI APTVTGNISI PTNFSMSIRT EYLQLYNTP VSVDCATYVC NGNSRCKQLL TQYTAACKTI ESALQLSARL ESVEVNSMLT ISEEALQLAT ISSFNGDGYN FTNVLGASVY DPASGRVVQ KRSVIEDLLF NKVVTNGLGT VDEDYKRCSN GRSVADLVCA QYYSGVMVLP GVVDAEKLHM YSASLIGGMA L GGITAAAA LPFSYAVQAR LNYLALQTDV LQRNQQLLAE SFNSAIGNIT SAFESVKEAI SQTSKGLNTV AHALTKVQEV VN SQGSALN QLTVQLQHNF QAISSSIDDI YSRLDILSAD VQVDRLITGR LSALNAFVAQ TLTKYTEVQA SRKLAQQKVN ECV KSQSQR YGFCGGDGEH IFSLVQAAPQ GLLFLHTVLV PGDFVNVLAI AGLCVNGEIA LTLREPGLVL FTHELQTYTA TEYF VSSRR MFEPRKPTVS DFVQIESCVV TYVNLTSDQL PDVIPDYIDV NKTLDEILAS LPNRTGPSLP LDVFNATYLN LTGEI ADLE QRSESLRNTT EELRSLINNI NNTLVDLEWL NRVETGSGYI PEAPRDGQAY VRKDGEWVLL STFLGRSLEV LFQGPG HHH HHHHHSAWSH PQFEKGGGSG GGGSGGSAWS HPQFEK

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 39 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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 in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.4.6)
Details: Final reconstruction calculated using non-uniform 3D refinement
Number images used: 112655
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: 3 / Avg.num./class: 49133 / Software - Name: cryoSPARC (ver. 2.4.6)
FSC plot (resolution estimation)

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