+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8265 | |||||||||
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Title | CIP-treated Brome Mosaic Virus (RNA2.3/4) | |||||||||
Map data | Cryo-EM density map of BMV (B2.3/4 CIP-treated) | |||||||||
Sample |
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Biological species | Brome mosaic virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.1 Å | |||||||||
Authors | Hoover HS / Wang JC-Y / Middleton S / Ni P / Zlotnick A / Vaughan RC / Kao CC | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8265.map.gz | 36.3 MB | EMDB map data format | |
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Header (meta data) | emd-8265-v30.xml emd-8265.xml | 13.3 KB 13.3 KB | Display Display | EMDB header |
Images | emd_8265.png | 52.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8265 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8265 | HTTPS FTP |
-Validation report
Summary document | emd_8265_validation.pdf.gz | 79 KB | Display | EMDB validaton report |
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Full document | emd_8265_full_validation.pdf.gz | 78.1 KB | Display | |
Data in XML | emd_8265_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8265 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8265 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8265.map.gz / Format: CCP4 / Size: 104 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM density map of BMV (B2.3/4 CIP-treated) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.512 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Brome mosaic virus
Entire | Name: Brome mosaic virus |
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Components |
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-Supramolecule #1: Brome mosaic virus
Supramolecule | Name: Brome mosaic virus / type: virus / ID: 1 / Parent: 0 / Details: BMV B2.3/4 CIP-treated / NCBI-ID: 12302 / Sci species name: Brome mosaic virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No |
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Host (natural) | Organism: Triticum aestivum (bread wheat) |
Molecular weight | Theoretical: 4.6 MDa |
Virus shell | Shell ID: 1 / Name: Capsid / Diameter: 280.0 Å / T number (triangulation number): 3 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 5.2 Details: 250 mM NaOAc and 10 mM MgCl2 (pH 5.2) + 10 units of calf intestinal phosphatase (CIP) |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK III |
Details | 0.5-1 mg/ml |
-Electron microscopy
Microscope | JEOL 3200FS |
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Temperature | Min: 97.0 K |
Specialist optics | Energy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.1 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 80000 |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
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