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- PDB-8x01: Structure of the Mumps Virus L Protein (state2) Bound by Phosphop... -

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Basic information

Entry
Database: PDB / ID: 8x01
TitleStructure of the Mumps Virus L Protein (state2) Bound by Phosphoprotein Tetramer
Components
  • Phosphoprotein
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Mumps virus polymerase complex / RNA-dependent RNA synthesis / Large protein / phosphoprotein.
Function / homology
Function and homology information


GDP polyribonucleotidyltransferase / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / ATP binding / cytoplasm
Similarity search - Function
RNA-directed RNA polymerase, paramyxovirus / P/V phosphoprotein, paramyxoviral / Paramyxovirus P/V phosphoprotein C-terminal / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesMumps orthorubulavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsLi, T.H. / Shen, Q.T.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Nat Commun / Year: 2024
Title: Structures of the mumps virus polymerase complex via cryo-electron microscopy.
Authors: Tianhao Li / Mingdong Liu / Zhanxi Gu / Xin Su / Yunhui Liu / Jinzhong Lin / Yu Zhang / Qing-Tao Shen /
Abstract: The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), ...The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L-P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.
History
DepositionNov 2, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: Phosphoprotein
C: Phosphoprotein
D: Phosphoprotein
E: Phosphoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)423,5687
Polymers423,4375
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 256833.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mumps orthorubulavirus / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: C0JJA4, RNA-directed RNA polymerase, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides, GDP polyribonucleotidyltransferase, Transferases; Transferring ...References: UniProt: C0JJA4, RNA-directed RNA polymerase, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides, GDP polyribonucleotidyltransferase, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein
Phosphoprotein


Mass: 41651.066 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mumps orthorubulavirus / Strain: Jeryl-Lynn / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: C0JJ97
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: The MuV polymerase complex of RNA-directed RNA polymerase L with tetrameric phosphoproteins
Type: COMPLEX
Details: The MuV polymerase complex expressed in Sf9 cells and purified by affinity chromatography and size-exlusive chromatography sequentially.
Entity ID: #2, #1 / Source: RECOMBINANT
Source (natural)Organism: Mumps orthorubulavirus / Strain: Jeryl-Lynn
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTRIS hydrochlorideTris-HCl1
36 mMmagnesium chlorideMgCl21
41 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.75 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 477568 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311010
ELECTRON MICROSCOPYf_angle_d0.52114946
ELECTRON MICROSCOPYf_dihedral_angle_d3.5021469
ELECTRON MICROSCOPYf_chiral_restr0.0391727
ELECTRON MICROSCOPYf_plane_restr0.0041891

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