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- EMDB-21957: Cryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotaviru... -

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Basic information

Entry
Database: EMDB / ID: EMD-21957
TitleCryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Reversed conformation
Map dataB-sharpened final map
Sample
  • Complex: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation
    • Protein or peptide: Outer capsid protein VP4
    • Protein or peptide: Intermediate capsid protein VP6
    • Protein or peptide: Outer capsid glycoprotein VP7
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / receptor-mediated virion attachment to host cell ...viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / receptor-mediated virion attachment to host cell / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / viral envelope / structural molecule activity / virion attachment to host cell / host cell plasma membrane / membrane / metal ion binding
Similarity search - Function
Rotavirus A/C, major capsid protein VP6 / Rotavirus major capsid protein VP6 / Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Virus capsid protein, alpha-helical / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Outer Capsid protein VP4 (Hemagglutinin) Concanavalin-like domain / Rotavirus VP4 helical domain ...Rotavirus A/C, major capsid protein VP6 / Rotavirus major capsid protein VP6 / Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Virus capsid protein, alpha-helical / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Outer Capsid protein VP4 (Hemagglutinin) Concanavalin-like domain / Rotavirus VP4 helical domain / Rotavirus VP4 helical domain / Outer capsid protein VP4 / Rotavirus VP4, membrane interaction domain superfamily / Rotavirus VP4, membrane interaction domain / Rotavirus VP4 membrane interaction domain / Viral capsid/haemagglutinin protein / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Intermediate capsid protein VP6 / Outer capsid protein VP4 / Outer capsid glycoprotein VP7
Similarity search - Component
Biological speciesRotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHerrmann T / Harrison SC / Jenni S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)CA-13202 United States
CitationJournal: Nature / Year: 2021
Title: Functional refolding of the penetration protein on a non-enveloped virus.
Authors: Tobias Herrmann / Raúl Torres / Eric N Salgado / Cristina Berciu / Daniel Stoddard / Daniela Nicastro / Simon Jenni / Stephen C Harrison /
Abstract: A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4. Here we ...A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.
History
DepositionMay 10, 2020-
Header (metadata) releaseJan 20, 2021-
Map releaseJan 20, 2021-
UpdateMar 10, 2021-
Current statusMar 10, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wxg
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6wxg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21957.png

Downloads & links

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Map

FileDownload / File: emd_21957.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-sharpened final map
Voxel sizeX=Y=Z: 1.231 Å
Density
Contour LevelBy EMDB: 0.05 / Movie #1: 0.028
Minimum - Maximum-0.09998287 - 0.15731071
Average (Standard dev.)5.620884e-06 (±0.010382091)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 393.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2311.2311.231
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z393.920393.920393.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1000.1570.000

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Supplemental data

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Mask #1

Fileemd_21957_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1

Fileemd_21957_half_map_1.map
AnnotationHalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map2

Fileemd_21957_half_map_2.map
AnnotationHalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rotavirus VP4, VP6, VP7 assembly in the foldback conformation

EntireName: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation
Components
  • Complex: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation
    • Protein or peptide: Outer capsid protein VP4
    • Protein or peptide: Intermediate capsid protein VP6
    • Protein or peptide: Outer capsid glycoprotein VP7
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation

SupramoleculeName: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Obtained from wild-type recoated rhesus rotavirus particles (wt rcTLP)
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Molecular weightTheoretical: 1.7 MDa

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Macromolecule #1: Outer capsid protein VP4

MacromoleculeName: Outer capsid protein VP4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Molecular weightTheoretical: 86.655586 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MASLIYRQLL TNSYTVDLSD EIQEIGSTKT QNVTINLGPF AQTGYAPVNW GPGETNDSTT VEPVLDGPYQ PTTFNPPVDY WMLLAPTAA GVVVEGTNNT DRWLATILVE PNVTSETRSY TLFGTQEQIT IANASQTQWK FIDVVKTTQN GSYSQYGPLQ S TPKLYAVM ...String:
MASLIYRQLL TNSYTVDLSD EIQEIGSTKT QNVTINLGPF AQTGYAPVNW GPGETNDSTT VEPVLDGPYQ PTTFNPPVDY WMLLAPTAA GVVVEGTNNT DRWLATILVE PNVTSETRSY TLFGTQEQIT IANASQTQWK FIDVVKTTQN GSYSQYGPLQ S TPKLYAVM KHNGKIYTYN GETPNVTTKY YSTTNYDSVN MTAFCDFYII PREEESTCTE YINNGLPPIQ NTRNIVPLAL SA RNIISHR AQANEDIVVS KTSLWKEMQY NRDITIRFKF ASSIVKSGGL GYKWSEISFK PANYQYTYTR DGEEVTAHTT CSV NGMNDF NFNGGSLPTD FVISRYEVIK ENSYVYVDYW DDSQAFRNMV YVRSLAANLN SVICTGGDYS FALPVGQWPV MTGG AVSLH SAGVTLSTQF TDFVSLNSLR FRFRLTVEEP SFSITRTRVS RLYGLPAANP NNGKEYYEVA GRFSLISLVP SNDDY QTPI TNSVTVRQDL ERQLGELREE FNALSQEIAM SQLIDLALLP LDMFSMFSGI KSTIDAAKSM ATSVMKKFKK SGLANS VST LTDSLSDAAS SISRGASIRS VGSSASAWTD VSTQITDVSS SVSSISTQTS TISRRLRLKE MATQTEGMNF DDISAAV LK TKIDRSTQIS PNTLPDIVTE ASEKFIPNRA YRVINNDEVF EAGTDGRFFA YRVETFDEIP FDVQKFADLV TDSPVISA I IDFKTLKNLN DNYGISRQQA FNLLRSDPRV LREFINQDNP IIRNRIEQLI MQCRL

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Macromolecule #2: Intermediate capsid protein VP6

MacromoleculeName: Intermediate capsid protein VP6 / type: protein_or_peptide / ID: 2 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Molecular weightTheoretical: 44.934766 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDVLYSLSKT LKDARDKIVE GTLYSNVSDL IQQFNQMIIT MNGNEFQTGG IGNLPIRNWN FDFGLLGTTL LNLDANYVET ARNTIDYFV DFVDNVCMDE MVRESQRNGI APQSDSLRKL SGIKFKRINF DNSSEYIENW NLQNRRQRTG FTFHKPNIFP Y SASFTLNR ...String:
MDVLYSLSKT LKDARDKIVE GTLYSNVSDL IQQFNQMIIT MNGNEFQTGG IGNLPIRNWN FDFGLLGTTL LNLDANYVET ARNTIDYFV DFVDNVCMDE MVRESQRNGI APQSDSLRKL SGIKFKRINF DNSSEYIENW NLQNRRQRTG FTFHKPNIFP Y SASFTLNR SQPAHDNLMG TMWLNAGSEI QVAGFDYSCA INAPANIQQF EHIVQLRRVL TTATITLLPD AERFSFPRVI NS ADGATTW YFNPVILRPN NVEVEFLLNG QIINTYQARF GTIIARNFDT IRLSFQLMRP PNMTPAVAAL FPNAQPFEHH ATV GLTLRI ESAVCESVLA DASKTMLANV TSVRQEYAIP VGPVFPPGMN WTDLITNYSP SREDNLQRVF TVASIRSMLV K

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Macromolecule #3: Outer capsid glycoprotein VP7

MacromoleculeName: Outer capsid glycoprotein VP7 / type: protein_or_peptide / ID: 3 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Molecular weightTheoretical: 37.136531 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MYGIEYTTVL TFLISLILLN YILKSLTRMM DFIIYRFLFI VVILSPLLKA QNYGINLPIT GSMDTAYANS TQEETFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTDIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM ...String:
MYGIEYTTVL TFLISLILLN YILKSLTRMM DFIIYRFLFI VVILSPLLKA QNYGINLPIT GSMDTAYANS TQEETFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTDIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM DITLYYYQQT DEANKWISMG SSCTIKVCPL NTQTLGIGCL TTDTATFEEV ATAEKLVITD VVDGVNHKLD VT TATCTIR NCKKLGPREN VAVIQVGGSD VLDITADPTT APQTERMMRI NWKKWWQVFY TVVDYVNQII QAMSKRSRSL NSA AFYYRI

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 18 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 54 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration1.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM2-Amino-2-(hydroxymethyl)propan-1,3-diolTris
100.0 mMNaClSodium chlorideSodium Chloride
1.0 mMCaCl2Calcium Chloride
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3 / Details: 4 ul sample volume, 4 sec blotting time.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 40605 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 27500
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3701 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 4107 / Average exposure time: 10.0 sec. / Average electron dose: 33.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14579 / Details: whole viral particles
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4v7q

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Details: Euler angle determination of viral particles by projection matching to cryo-EM map from pdb 4V7Q
Final 3D classificationNumber classes: 3 / Avg.num./class: 250000 / Software - Name: cisTEM
Details: Classification of VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM / Details: Refinement of euler angles for viral particles.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM
Details: Reconstruction final map after classification of VP4 sub-particles . VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)
Number images used: 252548
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

4v7q

,

1slq

)
Detailsphenix.real_space_refine
RefinementProtocol: OTHER
Output model

PDB-6wxg:
Cryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Reversed conformation

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