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- PDB-6wxg: Cryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotaviru... -

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Basic information

Entry
Database: PDB / ID: 6wxg
TitleCryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Reversed conformation
Components
  • Intermediate capsid protein VP6
  • Outer capsid glycoprotein VP7
  • Outer capsid protein VP4
KeywordsVIRAL PROTEIN / Complex / non-enveloped virus / viral particle / entry / membrane-penetration / rotavirus / VP4 / VP5* / VP8*
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHerrmann, T. / Harrison, S.C. / Jenni, S.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 20, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
1: Outer capsid protein VP4
2: Outer capsid protein VP4
3: Outer capsid protein VP4
A: Intermediate capsid protein VP6
B: Intermediate capsid protein VP6
C: Intermediate capsid protein VP6
D: Intermediate capsid protein VP6
E: Intermediate capsid protein VP6
F: Intermediate capsid protein VP6
G: Intermediate capsid protein VP6
H: Intermediate capsid protein VP6
I: Intermediate capsid protein VP6
J: Intermediate capsid protein VP6
K: Intermediate capsid protein VP6
L: Intermediate capsid protein VP6
M: Intermediate capsid protein VP6
N: Intermediate capsid protein VP6
O: Intermediate capsid protein VP6
P: Intermediate capsid protein VP6
Q: Intermediate capsid protein VP6
R: Intermediate capsid protein VP6
a: Outer capsid glycoprotein VP7
b: Outer capsid glycoprotein VP7
c: Outer capsid glycoprotein VP7
d: Outer capsid glycoprotein VP7
e: Outer capsid glycoprotein VP7
f: Outer capsid glycoprotein VP7
g: Outer capsid glycoprotein VP7
h: Outer capsid glycoprotein VP7
i: Outer capsid glycoprotein VP7
j: Outer capsid glycoprotein VP7
k: Outer capsid glycoprotein VP7
l: Outer capsid glycoprotein VP7
m: Outer capsid glycoprotein VP7
n: Outer capsid glycoprotein VP7
o: Outer capsid glycoprotein VP7
p: Outer capsid glycoprotein VP7
q: Outer capsid glycoprotein VP7
r: Outer capsid glycoprotein VP7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,743,396111
Polymers1,737,25039
Non-polymers6,14672
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Virus particles previously were verified to be assembled via various electron microscopy studies, infectivity assays and SDS PAGE analysis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer capsid protein VP4 / Hemagglutinin


Mass: 86655.586 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3] / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: G0YZG6
#2: Protein
Intermediate capsid protein VP6


Mass: 44934.766 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3] / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B2BN53
#3: Protein
Outer capsid glycoprotein VP7


Mass: 37136.531 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3] / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P12476
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 54 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rotavirus VP4, VP6, VP7 assembly in the foldback conformation
Type: COMPLEX
Details: Obtained from wild-type recoated rhesus rotavirus particles (wt rcTLP)
Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 1.7 MDa / Experimental value: NO
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris2-Amino-2-(hydroxymethyl)propan-1,3-diol1
2100 mMSodium ChlorideNaClSodium chloride1
31 mMCalcium ChlorideCaCl21
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298.15 K / Details: 4 ul sample volume, 4 sec blotting time

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Calibrated magnification: 40605 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 3000 nm / Cs: 2 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 33 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4107
Image scansWidth: 3838 / Height: 3701 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategoryDetails
1EMAN2particle selectione2boxer.py used to manually select viral particles
2SerialEMimage acquisition
4GctfCTF correction
7Omodel fitting
9PHENIX1.17.1-3660model refinement
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14579 / Details: whole viral particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252548 / Algorithm: FOURIER SPACE
Details: Reconstruction final map after classification of VP4 sub-particles . VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Details: phenix.real_space_refine
Atomic model building
IDPDB-ID 3D fitting-ID
14V7Q

4v7q

1
21SLQ

1slq

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 17.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0042103842
ELECTRON MICROSCOPYf_angle_d0.7514141487
ELECTRON MICROSCOPYf_chiral_restr0.046316259
ELECTRON MICROSCOPYf_plane_restr0.004418263
ELECTRON MICROSCOPYf_dihedral_angle_d13.143137691

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