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- EMDB-32338: Cryo-EM map of PEDV S protein with one protomer in the D0-up conf... -

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Basic information

Entry
Database: EMDB / ID: EMD-32338
TitleCryo-EM map of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-down conformation
Map dataCryo-EM map of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-up conformation.
Sample
  • Organelle or cellular component: Intact Porcine epidemic diarrhea virus (strain Pintung 52)
    • 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus ...Spike glycoprotein, Alphacoronavirus / Spike glycoprotein S1, coronavirus / Coronavirus spike glycoprotein S1 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesPorcine epidemic diarrhea virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.4 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
DepositionDec 3, 2021-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateDec 21, 2022-
Current statusDec 21, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32338.png

Downloads & links

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Map

FileDownload / File: emd_32338.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-up conformation.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 220 pix.
= 308. Å		1.4 Å/pix.
x 220 pix.
= 308. Å		1.4 Å/pix.
x 220 pix.
= 308. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.68718004 - 1.5117334
Average (Standard dev.)0.00012101826 (±0.06819567)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 308.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM half map (even) of PEDV S protein...

Fileemd_32338_half_map_1.map
AnnotationCryo-EM half map (even) of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-up conformation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map (odd) of PEDV S protein...

Fileemd_32338_half_map_2.map
AnnotationCryo-EM half map (odd) of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-up conformation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Intact Porcine epidemic diarrhea virus (strain Pintung 52)

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

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Supramolecule #1: Intact Porcine epidemic diarrhea virus (strain Pintung 52)

SupramoleculeName: Intact Porcine epidemic diarrhea virus (strain Pintung 52)
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The PEDV PT52 virus was propagated in Vero C1008 cells (ATCC No. CRL-1586) and then inactivated by 2% formaldehyde.
Source (natural)Organism: Porcine epidemic diarrhea virus / Strain: Pintung 52

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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
Molecular weightTheoretical: 152.005656 KDa
Recombinant expressionOrganism: Chlorocebus aethiops aethiops (mammal)
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 DDIYSRLDIL 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 KWPWWVWLII FIVLIFVVSL LVFCCISTGF CGCFGC CCA CFSGCCRGPR LQPYEVFEKV HVQ

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 19 / 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

BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMTristris(hydroxymethyl)aminomethane -
100.0 mMNaClsodium chloride
0.1 mMEDTAethylenediaminetetraacetic acid

Details: Blot for 3 seconds before plunging. Force 0.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe PEDV PT52 virus was propagated in Vero C1008 cells (ATCC No. CRL-1586) and then inactivated by 2% formaldehyde.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
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
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 / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
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: 337583
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vv5

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 9319
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.1) / Details: Non-uniform refinement in CryoSparc3.1
Final 3D classificationDetails: Non-uniform refinement in CryoSparc3.1
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7w73:
Cryo-EM map of PEDV S protein with one protomer in the D0-up conformation while the other two in the D0-down conformation

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