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- EMDB-33648: Symmetry-expanded and locally refined protomer structure of IPEC-... -

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Basic information

Entry
Database: EMDB / ID: EMD-33648
TitleSymmetry-expanded and locally refined protomer structure of IPEC-J2 cell-derived PEDV PT52 S with a CTD-close conformation
Map data
Sample
  • Organelle or cellular component: recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2P Spike
    • Protein or peptide: Spike glycoproteinSpike protein
  • 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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHsu STD / Draczkowski P / Wang YS
Funding support Taiwan, 6 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-CDA-109- L08 Taiwan
Academia Sinica (Taiwan)AS-IDR- 110-08 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 109-3114-Y-001-001 Taiwan
Ministry of Science and Technology (MoST, Taiwan)110-2113-M-001- 050-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)110-2311-B-001-013-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 110-2811-B-001-560 Taiwan
CitationJournal: Nat Commun / Year: 2022
Title: In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.
Authors: Cheng-Yu Huang / Piotr Draczkowski / Yong-Sheng Wang / Chia-Yu Chang / Yu-Chun Chien / Yun-Han Cheng / Yi-Min Wu / Chun-Hsiung Wang / Yuan-Chih Chang / Yen-Chen Chang / Tzu-Jing Yang / Yu-Xi ...Authors: Cheng-Yu Huang / Piotr Draczkowski / Yong-Sheng Wang / Chia-Yu Chang / Yu-Chun Chien / Yun-Han Cheng / Yi-Min Wu / Chun-Hsiung Wang / Yuan-Chih Chang / Yen-Chen Chang / Tzu-Jing Yang / Yu-Xi Tsai / Kay-Hooi Khoo / Hui-Wen Chang / Shang-Te Danny Hsu /
Abstract: Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, ...Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two different strains have been reported, but they reveal distinct N-terminal domain 0 (D0) architectures that may correspond to different functional states. The existence of the D0 is a unique feature of alphacoronavirus. Here we combined cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) to demonstrate in situ the asynchronous S protein D0 motions on intact viral particles of a highly virulent PEDV Pintung 52 strain. We further determined the cryo-EM structure of the recombinant S protein derived from a porcine cell line, which revealed additional domain motions likely associated with receptor binding. By integrating mass spectrometry and cryo-EM, we delineated the complex compositions and spatial distribution of the PEDV S protein N-glycans, and demonstrated the functional role of a key N-glycan in modulating the D0 conformation.
History
DepositionJun 21, 2022-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33648.png

Downloads & links

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Map

FileDownload / File: emd_33648.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.4444744 - 1.4586055
Average (Standard dev.)-0.00023053809 (±0.03921565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 385.83997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33648_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33648_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2...

EntireName: recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2P Spike
Components
  • Organelle or cellular component: recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2P Spike
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2...

SupramoleculeName: recombinant Porcine epidemic diarrhea virus (strain Pintung 52) 2P Spike
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Porcine epidemic diarrhea virus / Strain: Pintung 52
Recombinant expressionOrganism: Sus scrofa (pig) / Recombinant cell: IPEC-J2

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus
Molecular weightTheoretical: 153.739625 KDa
Recombinant expressionOrganism: Sus scrofa (pig)
SequenceString: MKSLTYFWLF LPVLSTLSLP QDVTRCSAKT NFRRFFSKFN VQAPAVVVLG GYLPIGENQG VNSTWYCAGQ HPTASGVHGI FVSHIRGGH GFEIGISQEP FDPSGYQLYL HKATNGNTNA TARLRICQFP SIKTLGPTAN NDVTIGRNCL FNKAIPAHMS E HSVVGITW ...String:
MKSLTYFWLF LPVLSTLSLP QDVTRCSAKT NFRRFFSKFN VQAPAVVVLG GYLPIGENQG VNSTWYCAGQ HPTASGVHGI FVSHIRGGH GFEIGISQEP FDPSGYQLYL HKATNGNTNA TARLRICQFP SIKTLGPTAN NDVTIGRNCL FNKAIPAHMS E HSVVGITW DNDRVTVFSD KIYYFYFKND WSRVATKCYN SGGCAMQYVY EPTYYMLNVT SAGEDGISYQ PCTANCIGYA AN VFATEPN GHIPEGFSFN NWFLLSNDST LVHGKVVSNQ PLLVNCLLAI PKIYGLGQFF SFNQTIDGVC NGAAVQRAPE ALR FNINDT SVILAEGSIV LHTALGTNFS FVCSNSSNPH LATFAIPLGA TQVPYYCFFK VDTYNSTVYK FLAVLPPTVR EIVI TKYGD VYVNGFGYLH LGLLDAVTIN FTGHGTDDDV SGFWTIASTN FVDALIEVQG TAIQRILYCD DPVSQLKCSQ VAFDL DDGF YPFSSRNLLS HEQPISFVTL PSFNAHSFVN ITVSASFGGH SGANLIASDT TINGFSSFCV DTRQFTISLS YNVTNS YGY VSNSQDSNCP FTLQSVNDYL SFSKFCVSTS LLASACTIDL FGYPEFGSGV KFTSLYFQFT KGELITGTPK PLEGVTD VS FMTLDVCTKY TIYGFKGEGI ITLTNSSFLA GVYYTSDSGQ LLAFKNVTSG AVYSVTPCSF SEQAAYVDDD IVGVISSL S SSTFNSTREL PGFFYHSNDG SNCTEPVLVY SNIGVCKSGS IGYVPSQSGQ VKIAPTVTGN ISIPTNFSMS IRTEYLQLY NTPVSVDCAT YVCNGNSRCK QLLTQYTAAC KTIESALQLS ARLESVEVNS MLTISEEALQ LATISSFNGD GYNFTNVLGV SVYDPARGR VVQKRSFIED LLFNKVVTNG LGTVDEDYKR CSNGRSVADL VCAQYYSGVM VLPGVVDAEK LHMYSASLIG G MVLGGFTA AAALPFSYAV QARLNYLALQ TDVLQRNQQL LAESFNSAIG NITSAFESVK EASSQTSRGL NTVAHALTKV QE VVNSQGA ALTQLTVQLQ HNFQAISSSI DDIYSRLDPP SADVQVDRLI TGRLSALNAF VAQTLTKYTE VQASRKLAQQ KVN ECVKSQ SQRYGFCGGD GEHIFSLVQA APQGLLFLHT VLVPSDFVDV IAIAGLCVND EIALTLREPG LVLFTHELQN HTAT EYFVS SRRMFEPRKP TVSDFVQIES CVVTYVNLTR DQLPDVIPDY IDVNKTRDEI LASLPNRTGP SLPLDVFNAT YLNLT GEIA DLEQRSESLR NTTEELQSLI YNINNTLVDL EWLNRVETYI KWPEFGSGGY IPEAPRDGQA YVRKDGEWVL LSTFLK GQD NSADIQHSGR PLESRGPFEQ KLISEEDLNM HTGHHHHHH

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 7 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

BufferpH: 7.6
Component:
ConcentrationFormulaName
50.0 mMTristris(hydroxymethyl)aminomethane -
150.0 mMNaClSodium chloridesodium chloride
0.02 %NaN3Sodium Azide

Details: Blot for 3 seconds before plunging. Force 0.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSymmetry-expanded and locally refined protomer structure of IPEC-J2 cell-derived PEDV PT52 S with a CTD-close conformation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3701 / Average electron dose: 55.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 337583
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vv5

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationDetails: Non-uniform refinement in CryoSparc3.1
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.1) / Details: Non-uniform refinement in CryoSparc3.1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 171864
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7y6u:
Symmetry-expanded and locally refined protomer structure of IPEC-J2 cell-derived PEDV PT52 S with a CTD-close conformation

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