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- EMDB-33704: Cryo-EM map of HEK293F cell-derived PEDV PT52 S T326I one D0-up a... -

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Basic information

Entry
Database: EMDB / ID: EMD-33704
TitleCryo-EM map of HEK293F cell-derived PEDV PT52 S T326I one D0-up and two D0-down
Map data
Sample
  • Organelle or cellular component: recombinant PEDV PT52 S protein expressed from HEK293F cells
    • Protein or peptide: recombinant PEDV PT52 S T326I expressed from HEK293F cells
Biological speciesPorcine epidemic diarrhea virus
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 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 27, 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_33704.png

Downloads & links

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Map

FileDownload / File: emd_33704.map.gz / Format: CCP4 / Size: 23 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 182 pix.
= 400.4 Å		2.2 Å/pix.
x 182 pix.
= 400.4 Å		2.2 Å/pix.
x 182 pix.
= 400.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.5217378 - 1.5260924
Average (Standard dev.)0.002551793 (±0.07835641)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions182182182
Spacing182182182
CellA=B=C: 400.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_33704_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33704_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : recombinant PEDV PT52 S protein expressed from HEK293F cells

EntireName: recombinant PEDV PT52 S protein expressed from HEK293F cells
Components
  • Organelle or cellular component: recombinant PEDV PT52 S protein expressed from HEK293F cells
    • Protein or peptide: recombinant PEDV PT52 S T326I expressed from HEK293F cells

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Supramolecule #1: recombinant PEDV PT52 S protein expressed from HEK293F cells

SupramoleculeName: recombinant PEDV PT52 S protein expressed from HEK293F cells
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Porcine epidemic diarrhea virus / Strain: Pintung 52
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Macromolecule #1: recombinant PEDV PT52 S T326I expressed from HEK293F cells

MacromoleculeName: recombinant PEDV PT52 S T326I expressed from HEK293F cells
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus / Strain: Pintung52
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKSLTYFWLF LPVLSTLSLP QDVTRCSAKT NFRRFFSKFN VQAPAVVVLG GYLPIGENQG VNSTWYCAGQ HPTASGVHGI FVSHIRGGHG FEIGISQEPF DPSGYQLYLH KATNGNTNAT ARLRICQFPS IKTLGPTANN DVTIGRNCLF NKAIPAHMSE HSVVGITWDN ...String:
MKSLTYFWLF LPVLSTLSLP QDVTRCSAKT NFRRFFSKFN VQAPAVVVLG GYLPIGENQG VNSTWYCAGQ HPTASGVHGI FVSHIRGGHG FEIGISQEPF DPSGYQLYLH KATNGNTNAT ARLRICQFPS IKTLGPTANN DVTIGRNCLF NKAIPAHMSE HSVVGITWDN DRVTVFSDKI YYFYFKNDWS RVATKCYNSG GCAMQYVYEP TYYMLNVTSA GEDGISYQPC TANCIGYAAN VFATEPNGHI PEGFSFNNWF LLSNDSTLVH GKVVSNQPLL VNCLLAIPKI YGLGQFFSFN QTIDGVCNGA AVQRAPEALR FNINDISVIL AEGSIVLHTA LGTNFSFVCS NSSNPHLATF AIPLGATQVP YYCFFKVDTY NSTVYKFLAV LPPTVREIVI TKYGDVYVNG FGYLHLGLLD AVTINFTGHG TDDDVSGFWT IASTNFVDAL IEVQGTAIQR ILYCDDPVSQ LKCSQVAFDL DDGFYPFSSR NLLSHEQPIS FVTLPSFNAH SFVNITVSAS FGGHSGANLI ASDTTINGFS SFCVDTRQFT ISLSYNVTNS YGYVSNSQDS NCPFTLQSVN DYLSFSKFCV STSLLASACT IDLFGYPEFG SGVKFTSLYF QFTKGELITG TPKPLEGVTD VSFMTLDVCT KYTIYGFKGE GIITLTNSSF LAGVYYTSDS GQLLAFKNVT SGAVYSVTPC SFSEQAAYVD DDIVGVISSL SSSTFNSTRE LPGFFYHSND GSNCTEPVLV YSNIGVCKSG SIGYVPSQSG QVKIAPTVTG NISIPTNFSM SIRTEYLQLY NTPVSVDCAT YVCNGNSRCK QLLTQYTAAC KTIESALQLS ARLESVEVNS MLTISEEALQ LATISSFNGD GYNFTNVLGV SVYDPARGRV VQKRSFIEDL LFNKVVTNGL GTVDEDYKRC SNGRSVADLV CAQYYSGVMV LPGVVDAEKL HMYSASLIGG MVLGGFTAAA ALPFSYAVQA RLNYLALQTD VLQRNQQLLA ESFNSAIGNI TSAFESVKEA SSQTSRGLNT VAHALTKVQE VVNSQGAALT QLTVQLQHNF QAISSSIDDI YSRLDPPSAD VQVDRLITGR LSALNAFVAQ TLTKYTEVQA SRKLAQQKVN ECVKSQSQRY GFCGGDGEHI FSLVQAAPQG LLFLHTVLVP SDFVDVIAIA GLCVNDEIAL TLREPGLVLF THELQNHTAT EYFVSSRRMF EPRKPTVSDF VQIESCVVTY VNLTRDQLPD VIPDYIDVNK TRDEILASLP NRTGPSLPLD VFNATYLNLT GEIADLEQRS ESLRNTTEEL QSLIYNINNT LVDLEWLNRV ETYIKWPEFG SGGYIPEAPR DGQAYVRKDG EWVLLSTFLK GQDNSADIQH SGRPLESRGP FEQKLISEED LNMHTGHHHH HH

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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 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
DetailsCryo-EM map of HEK293F cell-derived PEDV PT52 S protein with three D0-down

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
DetailsData were recorded with stage tilt at 0 and 30 degree
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2574 / Average electron dose: 50.0 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 / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 81000
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
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vv5

Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 15682
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

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