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- EMDB-8069: Prefusion structure of a human coronavirus spike protein -

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Basic information

Entry
Database: EMDB / ID: EMD-8069
TitlePrefusion structure of a human coronavirus spike protein
Map dataNone
Sample
  • Complex: HKU1 spike with attached foldon domain and mutated furin-cleavage site
    • Protein or peptide: Spike glycoprotein,Foldon chimera
Function / homology
Function and homology information


: / virion component / endocytosis involved in viral entry into host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / membrane => GO:0016020 / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / host cell plasma membrane ...: / virion component / endocytosis involved in viral entry into host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / membrane => GO:0016020 / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / host cell plasma membrane / virion membrane / identical protein binding
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, HKU1-like / : / Spike (S) protein S1 subunit, N-terminal domain, murine hepatitis virus-like / Fibritin C-terminal / Fibritin C-terminal region / Spike glycoprotein S2, coronavirus, C-terminal / Coronavirus spike glycoprotein S2, intravirion / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. ...Spike (S) protein S1 subunit, receptor-binding domain, HKU1-like / : / Spike (S) protein S1 subunit, N-terminal domain, murine hepatitis virus-like / Fibritin C-terminal / Fibritin C-terminal region / Spike glycoprotein S2, coronavirus, C-terminal / Coronavirus spike glycoprotein S2, intravirion / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / 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
Fibritin / Spike glycoprotein
Similarity search - Component
Biological speciesHuman coronavirus HKU1 (isolate N5) / Enterobacteria phage T4 (virus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 4.04 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 15, 2016-
Header (metadata) releaseMar 2, 2016-
Map releaseMar 2, 2016-
UpdateApr 22, 2020-
Current statusApr 22, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0335
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0335
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5i08
  • Surface level: 0.0335
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8069.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0335 / Movie #1: 0.0335
Minimum - Maximum-0.078518316 - 0.1497505
Average (Standard dev.)0.00004267100 (±0.005685221)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0790.1500.000

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Supplemental data

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Additional map: None

Fileemd_8069_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: None

Fileemd_8069_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: None

Fileemd_8069_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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: Spike glycoprotein,Foldon chimera

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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: Spike glycoprotein,Foldon chimera

MacromoleculeName: Spike glycoprotein,Foldon chimera / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Enterobacteria phage T4 (virus)
Molecular weightTheoretical: 144.295891 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VIGDFNCTNS FINDYNKTIP RISEDVVDVS LGLGTYYVLN RVYLNTTLLF TGYFPKSGAN FRDLALKGSI YLSTLWYKPP FLSDFNNGI FSKVKNTKLY VNNTLYSEFS TIVIGSVFVN TSYTIVVQPH NGILEITACQ YTMCEYPHTV CKSKGSIRNE S WHIDSSEP ...String:
VIGDFNCTNS FINDYNKTIP RISEDVVDVS LGLGTYYVLN RVYLNTTLLF TGYFPKSGAN FRDLALKGSI YLSTLWYKPP FLSDFNNGI FSKVKNTKLY VNNTLYSEFS TIVIGSVFVN TSYTIVVQPH NGILEITACQ YTMCEYPHTV CKSKGSIRNE S WHIDSSEP LCLFKKNFTY NVSADWLYFH FYQERGVFYA YYADVGMPTT FLFSLYLGTI LSHYYVMPLT CNAISSNTDN ET LEYWVTP LSRRQYLLNF DEHGVITNAV DCSSSFLSEI QCKTQSFAPN TGVYDLSGFT VKPVATVYRR IPNLPDCDID NWL NNVSVP SPLNWERRIF SNCNFNLSTL LRLVHVDSFS CNNLDKSKIF GSCFNSITVD KFAIPNRRRD DLQLGSSGFL QSSN YKIDI SSSSCQLYYS LPLVNVTINN FNPSSWNRRY GFGSFNLSSY DVVYSDHCFS VNSDFCPCAD PSVVNSCAKS KPPSA ICPA GTKYRHCDLD TTLYVKNWCR CSCLPDPIST YSPNTCPQKK VVVGIGEHCP GLGINEEKCG TQLNHSSCFC SPDAFL GWS FDSCISNNRC NIFSNFIFNG INSGTTCSND LLYSNTEIST GVCVNYDLYG ITGQGIFKEV SAAYYNNWQN LLYDSNG NI IGFKDFLTNK TYTILPCYSG RVSAAFYQNS SSPALLYRNL KCSYVLNNIS FISQPFYFDS YLGCVLNAVN LTSYSVSS C DLRMGSGFCI DYALPSSGGS GSGISSPYRF VTFEPFNVSF VNDSVETVGG LFEIQIPTNF TIAGHEEFIQ TSSPKVTID CSAFVCSNYA ACHDLLSEYG TFCDNINSIL NEVNDLLDIT QLQVANALMQ GVTLSSNLNT NLHSDVDNID FKSLLGCLGS QCGSSSRSL LEDLLFNKVK LSDVGFVEAY NNCTGGSEIR DLLCVQSFNG IKVLPPILSE TQISGYTTAA TVAAMFPPWS A AAGVPFSL NVQYRINGLG VTMDVLNKNQ KLIANAFNKA LLSIQNGFTA TNSALAKIQS VVNANAQALN SLLQQLFNKF GA ISSSLQE ILSRLDNLEA QVQIDRLING RLTALNAYVS QQLSDITLIK AGASRAIEKV NECVKSQSPR INFCGNGNHI LSL VQNAPY GLLFIHFSYK PTSFKTVLVS PGLCLSGDRG IAPKQGYFIK QNDSWMFTGS SYYYPEPISD KNVVFMNSCS VNFT KAPFI YLNNSIPNLS DFEAELSLWF KNHTSIAPNL TFNSHINATF LDLYYEMNVI QESIKSLNGS GYIPEAPRDG QAYVR KDGE WVLLSTFLGL EVLFQ

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
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: 3 uL sample was applied to grid for 30 seconds and then blotted. Grids were stained with 3 uL 1% uranyl formate for 60 seconds followed by blotting.
GridModel: EMS CF-2/2-4C C-Flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: 3 uL sample was applied to grid, blotted, and plunged into liquid ethane..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1049 / Average exposure time: 10.0 sec. / Average electron dose: 57.0 e/Å2
Details: Images were collected using Legionon and processed using Appion.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 39164
Details: 2188 particles were selected from a subset of the data using DoG Picker. These particles were used to generate a 3D model from which back projections were derived. Back projection images ...Details: 2188 particles were selected from a subset of the data using DoG Picker. These particles were used to generate a 3D model from which back projections were derived. Back projection images were used as templates for picking particles from the entire dataset using FindEM.
CTF correctionSoftware - Name: CTFFIND3
Startup modelType of model: INSILICO MODEL
In silico model: Initial model was generated with EMAN2 using a selection of 2D classifications from a subset of the data containing 2188 particles.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 629 / Avg.num./class: 50 / Software - Name: IMAGIC / Software - details: MSA
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4b1)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4b1) / Software - details: refine / Number images used: 31435
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel building and refinement were conducted using a combination of software programs.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 117 / Target criteria: EMRinger
Output model

PDB-5i08:
Prefusion structure of a human coronavirus spike protein

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