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- EMDB-8264: Mock-treated Brome Mosaic Virus (RNA2.3/4) -

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Basic information

Entry
Database: EMDB / ID: EMD-8264
TitleMock-treated Brome Mosaic Virus (RNA2.3/4)
Map dataCryo-EM density map of BMV (B2.3/4 Mock-treated)
Sample
  • Virus: Brome mosaic virus
Biological speciesBrome mosaic virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsHoover HS / Wang JC-Y / Middleton S / Ni P / Zlotnick A / Vaughan RC / Kao CC
CitationJournal: J Virol / Year: 2016
Title: Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection.
Authors: Haley S Hoover / Joseph Che-Yen Wang / Stefani Middleton / Peng Ni / Adam Zlotnick / Robert C Vaughan / C Cheng Kao /
Abstract: The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV ...The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.
IMPORTANCE: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene ...IMPORTANCE: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.
History
DepositionJun 25, 2016-
Header (metadata) releaseJul 6, 2016-
Map releaseJul 6, 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: 7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8264.map.gz / Format: CCP4 / Size: 104 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM density map of BMV (B2.3/4 Mock-treated)
Voxel sizeX=Y=Z: 1.512 Å
Density
Contour LevelBy AUTHOR: 7. / Movie #1: 7
Minimum - Maximum-14.736412 - 33.876938000000003
Average (Standard dev.)0.9484691 (±4.480711)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions301301301
Spacing301301301
CellA=B=C: 455.112 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5121.5121.512
M x/y/z301301301
origin x/y/z0.0000.0000.000
length x/y/z455.112455.112455.112
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS301301301
D min/max/mean-14.73633.8770.948

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Supplemental data

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Sample components

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Entire : Brome mosaic virus

EntireName: Brome mosaic virus
Components
  • Virus: Brome mosaic virus

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Supramolecule #1: Brome mosaic virus

SupramoleculeName: Brome mosaic virus / type: virus / ID: 1 / Parent: 0 / Details: BMV B2.3/4 Mock-treated / NCBI-ID: 12302 / Sci species name: Brome mosaic virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Triticum aestivum (bread wheat)
Molecular weightTheoretical: 4.6 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 280.0 Å / T number (triangulation number): 3

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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

Concentration0.5 mg/mL
BufferpH: 5.2 / Details: 250 mM NaOAc and 10 mM MgCl2 (pH 5.2)
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK III
Details0.5-1 mg/ml

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Electron microscopy

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.1 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 80000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 97.0 K
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.0 µm / Average exposure time: 0.5 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Particle selectionNumber selected: 32214
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: OTHER / Details: de novo model building in AUTO3DEM
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: Auto3DEM (ver. 4.04)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: Auto3DEM (ver. 4.04)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Auto3DEM (ver. 4.04) / Number images used: 19332

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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