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- EMDB-8067: HKU1 spike with attached foldon domain and wild-type furin-cleava... -

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Basic information

Entry
Database: EMDB / ID: EMD-8067
TitleHKU1 spike with attached foldon domain and wild-type furin-cleavage site
Map dataNone
Sample
  • Complex: HKU1 spike with attached foldon domain and mutated furin-cleavage site
    • Protein or peptide: HKU1 spike with attached foldon and a mutated furin-cleavage site
Biological speciesHuman coronavirus HKU1 (isolate N5)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsKirchdoerfer RN / Cottrell CA / Wang N / Pallesen J / Yassine HM / Turner HL / Corbett KS / Graham BS / McLellan JS / Ward AB
CitationJournal: Nature / Year: 2016
Title: Pre-fusion structure of a human coronavirus spike protein.
Authors: Robert N Kirchdoerfer / Christopher A Cottrell / Nianshuang Wang / Jesper Pallesen / Hadi M Yassine / Hannah L Turner / Kizzmekia S Corbett / Barney S Graham / Jason S McLellan / Andrew B Ward /
Abstract: HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic ...HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.
History
DepositionFeb 2, 2016-
Header (metadata) releaseFeb 24, 2016-
Map releaseMar 9, 2016-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.79
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.79
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8067.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 4.79 / Movie #1: 4.79
Minimum - Maximum-7.015168 - 12.855499999999999
Average (Standard dev.)-0.000000012143606 (±0.8098088)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 393.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-7.01512.856-0.000

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Supplemental data

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Sample components

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Entire : HKU1 spike with attached foldon domain and mutated furin-cleavage site

EntireName: HKU1 spike with attached foldon domain and mutated furin-cleavage site
Components
  • Complex: HKU1 spike with attached foldon domain and mutated furin-cleavage site
    • Protein or peptide: HKU1 spike with attached foldon and a mutated furin-cleavage site

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Supramolecule #1: HKU1 spike with attached foldon domain and mutated furin-cleavage site

SupramoleculeName: HKU1 spike with attached foldon domain and mutated furin-cleavage site
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human coronavirus HKU1 (isolate N5)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: FreeStyle 293F / Recombinant plasmid: pVRC8400
Molecular weightTheoretical: 420 KDa

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Macromolecule #1: HKU1 spike with attached foldon and a mutated furin-cleavage site

MacromoleculeName: HKU1 spike with attached foldon and a mutated furin-cleavage site
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human coronavirus HKU1 (isolate N5)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VIGDFNCTNS FINDYNKTIP RISEDVVDVS LGLGTYYVLN RVYLNTTLLF TGYFPKSGAN FRDLALKGSI YLSTLWYKPP FLSDFNNGIF SKVKNTKLYV NNTLYSEFST IVIGSVFVNT SYTIVVQPHN GILEITACQY TMCEYPHTVC KSKGSIRNES WHIDSSEPLC ...String:
VIGDFNCTNS FINDYNKTIP RISEDVVDVS LGLGTYYVLN RVYLNTTLLF TGYFPKSGAN FRDLALKGSI YLSTLWYKPP FLSDFNNGIF SKVKNTKLYV NNTLYSEFST IVIGSVFVNT SYTIVVQPHN GILEITACQY TMCEYPHTVC KSKGSIRNES WHIDSSEPLC LFKKNFTYNV SADWLYFHFY QERGVFYAYY ADVGMPTTFL FSLYLGTILS HYYVMPLTCN AISSNTDNET LEYWVTPLSR RQYLLNFDEH GVITNAVDCS SSFLSEIQCK TQSFAPNTGV YDLSGFTVKP VATVYRRIPN LPDCDIDNWL NNVSVPSPLN WERRIFSNCN FNLSTLLRLV HVDSFSCNNL DKSKIFGSCF NSITVDKFAI PNRRRDDLQL GSSGFLQSSN YKIDISSSSC QLYYSLPLVN VTINNFNPSS WNRRYGFGSF NLSSYDVVYS DHCFSVNSDF CPCADPSVVN SCAKSKPPSA ICPAGTKYRH CDLDTTLYVK NWCRCSCLPD PISTYSPNTC PQKKVVVGIG EHCPGLGINE EKCGTQLNHS SCFCSPDAFL GWSFDSCISN NRCNIFSNFI FNGINSGTTC SNDLLYSNTE ISTGVCVNYD LYGITGQGIF KEVSAAYYNN WQNLLYDSNG NIIGFKDFLT NKTYTILPCY SGRVSAAFYQ NSSSPALLYR NLKCSYVLNN ISFISQPFYF DSYLGCVLNA VNLTSYSVSS CDLRMGSGFC IDYALPSSGG SGSGISSPYR FVTFEPFNVS FVNDSVETVG GLFEIQIPTN FTIAGHEEFI QTSSPKVTID CSAFVCSNYA ACHDLLSEYG TFCDNINSIL NEVNDLLDIT QLQVANALMQ GVTLSSNLNT NLHSDVDNID FKSLLGCLGS QCGSSSRSLL EDLLFNKVKL SDVGFVEAYN NCTGGSEIRD LLCVQSFNGI KVLPPILSET QISGYTTAAT VAAMFPPWSA AAGVPFSLNV QYRINGLGVT MDVLNKNQKL IANAFNKALL SIQNGFTATN SALAKIQSVV NANAQALNSL LQQLFNKFGA ISSSLQEILS RLDNLEAQVQ IDRLINGRLT ALNAYVSQQL SDITLIKAGA SRAIEKVNEC VKSQSPRINF CGNGNHILSL VQNAPYGLLF IHFSYKPTSF KTVLVSPGLC LSGDRGIAPK QGYFIKQNDS WMFTGSSYYY PEPISDKNVV FMNSCSVNFT KAPFIYLNNS IPNLSDFEAE LSLWFKNHTS IAPNLTFNSH INATFLDLYY EMNVIQESIK SLNGSGYIPE APRDGQAYVR KDGEWVLLST FLGLEVLFQ

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.27 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
2.0 mMC4H11NO3Tris
StainingType: NEGATIVE / Material: uranyl formate
Details: 3uL sample was applied to grid for 30 seconds and then blotted. Grids were stained with 3uL 1% uranyl formate for 60 seconds followed by blotting.
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Number real images: 175 / Average exposure time: 1.0 sec. / Average electron dose: 28.0 e/Å2
Details: Images were collected using Legionon and processed using Appion.
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6536
Startup modelType of model: INSILICO MODEL
In silico model: Initial model was generated with EMAN2 using a selection of 2D classifications.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2
Final 3D classificationNumber classes: 106 / Avg.num./class: 50 / Software - Name: IMAGIC / Software - details: MRA
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionNumber classes used: 106 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1 / Software - details: refine / Number images used: 5281

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