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- EMDB-32332: Subtomogram averaging of PEDV (Pintung 52) S protein with all thr... -

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Basic information

Entry
Database: EMDB / ID: EMD-32332
TitleSubtomogram averaging of PEDV (Pintung 52) S protein with all three protomers in the D0-down conformation determined in situ on intact viral particles.
Map data
Sample
  • Organelle or cellular component: Intact Porcine epidemic diarrhea virus (strain Pintung 52)
KeywordsPEDV / Spike / Glycoprotein / VIRAL PROTEIN
Biological speciesPorcine epidemic diarrhea virus
Methodsubtomogram averaging / cryo EM / Resolution: 19.0 Å
AuthorsHsu STD / Draczkowski P / Wang YS / Huang CY
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)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)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 2, 2021-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32332.png

Downloads & links

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Map

FileDownload / File: emd_32332.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.56 Å/pix.
x 64 pix.
= 355.712 Å		5.56 Å/pix.
x 64 pix.
= 355.712 Å		5.56 Å/pix.
x 64 pix.
= 355.712 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.558 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.01
Minimum - Maximum-5.335215 - 9.245336999999999
Average (Standard dev.)0.0069802906 (±0.48979345)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 355.712 Å
α=β=γ: 90.0 °

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Supplemental data

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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)

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
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 R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 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
DetailsTomography 4 Software (Thermo Fisher Scientific) was used for tilt series acquisition
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 2.7 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: 6.0 µm / Nominal defocus min: 1.5 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

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: Dynamo (ver. 1.1.514), RELION (ver. 3.1)) / Number subtomograms used: 2385
ExtractionNumber tomograms: 90 / Number images used: 15065
Software: (Name: Dynamo (ver. 1.1.514), UCSF Chimera (ver. 1.13), MATLAB (ver. R2020a))
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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