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- PDB-5h0r: RNA dependent RNA polymerase ,vp4,dsRNA -

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Basic information

Entry
Database: PDB / ID: 5h0r
TitleRNA dependent RNA polymerase ,vp4,dsRNA
Components
  • (RNA (42-MER)) x 2
  • RNA-dependent RNA polymerase
  • VP4 protein
KeywordsTRANSFERASE/RNA / structural classification / TRANSFERASE-RNA complex
Function / homology
Function and homology information


viral genome replication / RNA-dependent RNA polymerase activity / RNA binding
Similarity search - Function
: / : / CPV, RNA-directed RNA polymerase, N-terminal / CPV, RNA-directed RNA polymerase, central polymerase domain / CPV, RNA-directed RNA polymerase, C-terminal / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA-dependent RNA polymerase / VP4 protein
Similarity search - Component
Biological speciesBombyx mori cytoplasmic polyhedrosis virus
Dendrolimus punctatus cypovirus 1
Cypovirus (cytoplasmic polyhedrosis viruses)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLi, X. / Zhou, N. / Chen, W. / Zhu, B. / Wang, X. / Xu, B. / Wang, J. / Liu, H. / Cheng, L.
Funding support China, 5items
OrganizationGrant numberCountry
the National Research and Development Program of China2016YFA0501103 China
he National Research and Development Program of China2015CB910104 China
the National Natural Science Foundation of China91530321 China
the National Natural Science Foundation of China31570727 China
the National Natural Science Foundation of China31570742 China
CitationJournal: J Mol Biol / Year: 2017
Title: Near-Atomic Resolution Structure Determination of a Cypovirus Capsid and Polymerase Complex Using Cryo-EM at 200kV.
Authors: Xiaowu Li / Niyun Zhou / Wenyuan Chen / Bin Zhu / Xurong Wang / Bin Xu / Jiawei Wang / Hongrong Liu / Lingpeng Cheng /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) allows the high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than ...Single-particle cryo-electron microscopy (cryo-EM) allows the high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5Å) cryo-EM structures reported to date were obtained by using 300kV transmission electron microscopes (TEMs). We report here the structures of a cypovirus capsid of 750-Å diameter at 3.3-Å resolution and of RNA-dependent RNA polymerase (RdRp) complexes within the capsid at 3.9-Å resolution using a 200-kV TEM. The newly resolved structure revealed conformational changes of two subdomains in the RdRp. These conformational changes, which were involved in RdRp's switch from non-transcribing to transcribing mode, suggest that the RdRp may facilitate the unwinding of genomic double-stranded RNA. The possibility of 3-Å resolution structural determinations for biological assemblies of relatively small sizes using cryo-EM at 200kV was discussed.
History
DepositionOct 6, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 25, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_image_scans
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
F: RNA-dependent RNA polymerase
G: VP4 protein
H: RNA (42-MER)
I: RNA (42-MER)


Theoretical massNumber of molelcules
Total (without water)229,4084
Polymers229,4084
Non-polymers00
Water00
1
F: RNA-dependent RNA polymerase
H: RNA (42-MER)
I: RNA (42-MER)


Theoretical massNumber of molelcules
Total (without water)165,6733
Polymers165,6733
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
G: VP4 protein


Theoretical massNumber of molelcules
Total (without water)63,7351
Polymers63,7351
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA-dependent RNA polymerase


Mass: 139077.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori cytoplasmic polyhedrosis virus
Gene: RdRp
Production host: Cypovirus (cytoplasmic polyhedrosis viruses)
References: UniProt: A0A0S1LIW6
#2: Protein VP4 protein


Mass: 63734.707 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dendrolimus punctatus cypovirus 1
Production host: Cypovirus (cytoplasmic polyhedrosis viruses)
References: UniProt: Q80A92
#3: RNA chain RNA (42-MER)


Mass: 13781.683 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Cypovirus (cytoplasmic polyhedrosis viruses)
#4: RNA chain RNA (42-MER)


Mass: 12814.002 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Cypovirus (cytoplasmic polyhedrosis viruses)

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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

Component
IDNameTypeEntity IDParent-IDSource
1RNA dependent RNA polymerase ,VP4,dsRNACOMPLEXall0MULTIPLE SOURCES
2RNA dependent RNA polymeraseCOMPLEX#11RECOMBINANT
3VP4COMPLEX#21RECOMBINANT
4dsRNACOMPLEX#31MULTIPLE SOURCES
5dsRNACOMPLEX#41MULTIPLE SOURCES
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Cypovirus (cytoplasmic polyhedrosis viruses)10981
22Cypovirus (cytoplasmic polyhedrosis viruses)10981
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 20 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0215526
ELECTRON MICROSCOPYf_angle_d0.7921397
ELECTRON MICROSCOPYf_dihedral_angle_d8.47312170
ELECTRON MICROSCOPYf_chiral_restr0.0472465
ELECTRON MICROSCOPYf_plane_restr0.0042425

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