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- PDB-3j6q: Identification of the active sites in the methyltransferases of a... -

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Basic information

Entry
Database: PDB / ID: 3j6q
TitleIdentification of the active sites in the methyltransferases of a transcribing dsRNA virus
ComponentsStructural protein VP3Structure
KeywordsVIRUS / dsRNA virus / Reoviridae / RNA capping / RNA methyltransferase
Function / homology
Function and homology information


: / : / : / : / Reovirus VP3 protein, guanylyltransferase (GTase) / Reovirus turret protein, bridge domain / Reovirus VP3 protein, Methyltransferase domain 1 / Reovirus VP3 protein, Methyltransferase domain 2
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / Structural protein VP3
Similarity search - Component
Biological speciesBombyx mori cypovirus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhu, B. / Yang, C. / Liu, H. / Cheng, L. / Song, F. / Zeng, S. / Huang, X. / Ji, G. / Zhu, P.
CitationJournal: J Mol Biol / Year: 2014
Title: Identification of the active sites in the methyltransferases of a transcribing dsRNA virus.
Authors: Bin Zhu / Chongwen Yang / Hongrong Liu / Lingpeng Cheng / Feng Song / Songjun Zeng / Xiaojun Huang / Gang Ji / Ping Zhu /
Abstract: Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae ...Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2'-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-L-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.
History
DepositionMar 20, 2014Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Oct 8, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 22, 2014Group: Other
Revision 1.2Dec 18, 2019Group: Data collection / Database references / Derived calculations
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_software / struct_conn
Item: _em_software.image_processing_id / _struct_conn.pdbx_leaving_atom_flag

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Assembly

Deposited unit
A: Structural protein VP3
B: Structural protein VP3
C: Structural protein VP3
D: Structural protein VP3
E: Structural protein VP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)606,29415
Polymers602,4505
Non-polymers3,84410
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Structural protein VP3 / Structure


Mass: 120489.984 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Bombyx mori cypovirus 1 / References: UniProt: Q914N6
#2: Chemical
ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C14H20N6O5S

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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: transcribing cypovirus / Type: VIRUS
Details of virusEmpty: NO / Enveloped: NO / Host category: VERTEBRATES / Isolate: OTHER / Type: VIRION
Natural hostOrganism: Homo sapiens
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 10, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Calibrated magnification: 125390 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Processing

EM software
IDNameCategory
1EMAN3D reconstruction
2IMIRS3D reconstruction
3ISAF3D reconstruction
CTF correctionDetails: EMAN
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Cross-common lines / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30000 / Details: Applied symmetry: C1 / Symmetry type: POINT
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms42315 0 260 0 42575

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