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- PDB-7yfe: In situ structure of polymerase complex of mammalian reovirus in ... -

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Basic information

Entry
Database: PDB / ID: 7yfe
TitleIn situ structure of polymerase complex of mammalian reovirus in virion
Components
  • Lambda-2 protein
  • Mu-2 protein
  • RNA (5'-R(P*AP*CP*GP*AP*UP*UP*AP*GP*C)-3')
  • RNA (5'-R(P*AP*UP*CP*GP*U)-3')
  • RNA (5'-R(P*GP*CP*U)-3')
  • RNA helicase
  • RNA-directed RNA polymerase
KeywordsVIRAL PROTEIN/RNA / mammalian reovirus / cryo-em / RNA dependent RNA polymerase / transcription / VIRAL PROTEIN / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


viral inner capsid / host cytoskeleton / viral outer capsid / 7-methylguanosine mRNA capping / viral genome replication / mRNA guanylyltransferase activity / viral capsid / viral nucleocapsid / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm ...viral inner capsid / host cytoskeleton / viral outer capsid / 7-methylguanosine mRNA capping / viral genome replication / mRNA guanylyltransferase activity / viral capsid / viral nucleocapsid / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA helicase activity / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTP binding / structural molecule activity / RNA binding / ATP binding
Similarity search - Function
Reovirus minor core protein, Mu-2 / Reovirus minor core protein Mu-2 / : / : / : / : / : / : / : / : ...Reovirus minor core protein, Mu-2 / Reovirus minor core protein Mu-2 / : / : / : / : / : / : / : / : / Reovirus core-spike protein lambda-2 (L2), 6th domain / Reovirus core-spike protein lambda-2 (L2), 7th domain / Reovirus core-spike protein lambda-2 (L2), ferredoxin-like domain / Reovirus core-spike protein lambda-2 (L2), GTase domain / Reovirus core-spike protein lambda-2 (L2), N-terminal / Reovirus core-spike protein lambda-2 (L2), methyltransferase-1 / Reovirus core-spike protein lambda-2 (L2), methyltransferase-2 / : / Inner capsid protein lambda-1/VP3 / Reovirus core-spike lambda-2 / Reovirus RNA-dependent RNA polymerase lambda 3 / Reovirus core-spike protein lambda-2 (L2), C-terminal / Reovirus RNA-dependent RNA polymerase lambda 3 / Inner capsid protein lambda-1/ VP3 / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Immunoglobulin-like fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA / RNA-directed RNA polymerase / Lambda-2 protein / Mu-2 protein / Lambda-1 protein
Similarity search - Component
Biological speciesMammalian orthoreovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBao, K.Y. / Zhang, X.L. / Li, D.Y. / Zhu, P.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31730023 China
National Natural Science Foundation of China (NSFC)31521002 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: In situ structures of polymerase complex of mammalian reovirus illuminate RdRp activation and transcription regulation.
Authors: Keyan Bao / Xueli Zhang / Dongyu Li / Wei Sun / Zhenzhao Sun / Jingfei Wang / Ping Zhu /
Abstract: Mammalian reovirus (reovirus) is a multilayered, turreted member of characterized by transcription of dsRNA genome within the innermost capsid shell. Here, we present high-resolution in situ ...Mammalian reovirus (reovirus) is a multilayered, turreted member of characterized by transcription of dsRNA genome within the innermost capsid shell. Here, we present high-resolution in situ structures of reovirus transcriptase complex in an intact double-layered virion, and in the uncoated single-layered core particles in the unloaded, reloaded, pre-elongation, and elongation states, respectively, obtained by cryo-electron microscopy and sub-particle reconstructions. At the template entry of RNA-dependent RNA polymerase (RdRp), the RNA-loading region gets flexible after uncoating resulting in the unloading of terminal genomic RNA and inactivity of transcription. However, upon adding transcriptional substrates, the RNA-loading region is recovered leading the RNAs loaded again. The priming loop in RdRp was found to play a critical role in regulating transcription, which hinders the elongation of transcript in virion and triggers the rearrangement of RdRp C-terminal domain (CTD) during elongation, resulting in splitting of template-transcript hybrid and opening of transcript exit. With the integration of these structures, a transcriptional model of reovirus with five states is proposed. Our structures illuminate the RdRp activation and regulation of the multilayered turreted reovirus.
History
DepositionJul 8, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: RNA helicase
2: RNA helicase
3: RNA helicase
4: RNA helicase
5: RNA helicase
A: RNA helicase
B: RNA helicase
C: RNA helicase
D: RNA helicase
E: RNA helicase
H: Lambda-2 protein
I: Lambda-2 protein
J: Lambda-2 protein
K: Lambda-2 protein
L: Lambda-2 protein
M: RNA (5'-R(P*GP*CP*U)-3')
N: RNA (5'-R(P*AP*UP*CP*GP*U)-3')
R: RNA-directed RNA polymerase
T: RNA (5'-R(P*AP*CP*GP*AP*UP*UP*AP*GP*C)-3')
U: Mu-2 protein
a: RNA helicase
b: RNA helicase
c: RNA helicase
d: RNA helicase
e: RNA helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)3,078,51632
Polymers3,078,05825
Non-polymers4587
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 22 molecules 12345ABCDEabcdeHIJKLRU

#1: Protein
RNA helicase / Lambda 1


Mass: 141801.297 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 / References: UniProt: C9E874, RNA helicase
#2: Protein
Lambda-2 protein


Mass: 143963.438 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 / References: UniProt: C9E871
#5: Protein RNA-directed RNA polymerase / RdRp Lambda 3


Mass: 142472.953 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 / References: UniProt: C9E870, RNA-directed RNA polymerase
#7: Protein Mu-2 protein


Mass: 83434.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 / References: UniProt: C9E872

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RNA chain , 3 types, 3 molecules MNT

#3: RNA chain RNA (5'-R(P*GP*CP*U)-3')


Mass: 911.596 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Transcript RNA / Source: (synth.) Mammalian orthoreovirus 3
#4: RNA chain RNA (5'-R(P*AP*UP*CP*GP*U)-3')


Mass: 1546.968 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Encoding RNA / Source: (synth.) Mammalian orthoreovirus 3
#6: RNA chain RNA (5'-R(P*AP*CP*GP*AP*UP*UP*AP*GP*C)-3')


Mass: 2855.767 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Template RNA / Source: (synth.) Mammalian orthoreovirus 3

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Non-polymers , 1 types, 7 molecules

#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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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
1Mammalian orthoreovirus 3COMPLEX#5-#6, #3-#4, #7, #1-#20MULTIPLE SOURCES
2Mammalian orthoreovirus 3VIRUS#5, #7, #1-#21NATURAL
3RNACOMPLEX#6, #3-#41SYNTHETIC
Source (natural)Organism: Mammalian orthoreovirus 3
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50031 / Symmetry type: POINT

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