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- PDB-6u1x: Structure of the Vesicular Stomatitis Virus L Protein in Complex ... -

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Basic information

Entry
Database: PDB / ID: 6u1x
TitleStructure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor (3.0 A resolution)
Components
  • Phosphoprotein
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / Vesicular stomatitis virus / RNA-dependent RNA polymerase / L protein / P protein / phosphoprotein / single particle analysis / transcription / replication / virus / viral / RdRp / PRNTase / nonsegmented negative-sense RNA viruses
Function / homology
Function and homology information


NNS virus cap methyltransferase / RNA folding chaperone / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / viral transcription / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral genome replication / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm ...NNS virus cap methyltransferase / RNA folding chaperone / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / viral transcription / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral genome replication / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / phosphorylation / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding
Similarity search - Function
: / Virus-capping methyltransferase, C-terminal / : / Vesiculovirus phosphoprotein / Phosphoprotein, central domain / : / Virus-capping methyltransferase, connector domain / Phosphoprotein, C-terminal domain, viral / RNA-directed RNA polymerase, rhabdovirus / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus ...: / Virus-capping methyltransferase, C-terminal / : / Vesiculovirus phosphoprotein / Phosphoprotein, central domain / : / Virus-capping methyltransferase, connector domain / Phosphoprotein, C-terminal domain, viral / RNA-directed RNA polymerase, rhabdovirus / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / 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. / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesVesicular stomatitis Indiana virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsJenni, S. / Bloyet, L.M. / Dias-Avalos, R. / Liang, B. / Wheelman, S.P.J. / Grigorieff, N. / Harrison, S.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R37 AI059371 United States
CitationJournal: Cell Rep / Year: 2020
Title: Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor.
Authors: Simon Jenni / Louis-Marie Bloyet / Ruben Diaz-Avalos / Bo Liang / Sean P J Whelan / Nikolaus Grigorieff / Stephen C Harrison /
Abstract: The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A ...The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 Å resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with an RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5' nucleotide.
History
DepositionAug 17, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
P: Phosphoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)271,3874
Polymers271,2562
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3280 Å2
ΔGint-15 kcal/mol
Surface area80130 Å2

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Components

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


Mass: 241313.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vesicular stomatitis Indiana virus (strain San Juan)
Strain: San Juan / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P03523, RNA-directed RNA polymerase, mRNA (guanine-N7)-methyltransferase, GDP polyribonucleotidyltransferase, EC: 2.1.1.296
#2: Protein Phosphoprotein / P protein / Protein M1


Mass: 29941.982 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vesicular stomatitis Indiana virus (strain San Juan)
Strain: San Juan / Production host: Escherichia coli (E. coli) / References: UniProt: P03520
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: VSV L:P / Type: COMPLEX
Details: L protein (full-length) in complex with P protein (35-106)
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Vesicular stomatitis virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
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
Image recordingAverage exposure time: 5 sec. / Electron dose: 100 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 3307
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategoryDetails
4CTFFIND4CTF correctionPart of cisTEM
7O14.0.0model fitting
9FREALIGNXinitial Euler assignmentPart of cisTEM
10FREALIGNXfinal Euler assignmentPart of cisTEM
11FREALIGNXclassificationPart of cisTEM
12FREALIGNX3D reconstructionPart of cisTEM
13PHENIX1.16-3549model refinement
Image processingDetails: Movies were gain-corrected and aligned using cisTEM.
CTF correctionDetails: CTF correction using cisTEM / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 246825 / Details: Resolution limit for picking: 2 nm
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83979 / Algorithm: FOURIER SPACE
Details: Refinement and classification were limited to a resolution of 0.4 nm to produce unbiased resolution estimate at final estimate resolution.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 70.6 / Protocol: OTHER / Space: REAL / Target criteria: CC + restraints
Atomic model buildingPDB-ID: 5A22

5a22


Pdb chain-ID: A / Accession code: 5A22 / Pdb chain residue range: 35-2109 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 70.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003233923
ELECTRON MICROSCOPYf_angle_d0.567461335
ELECTRON MICROSCOPYf_chiral_restr0.03932561
ELECTRON MICROSCOPYf_plane_restr0.00334933
ELECTRON MICROSCOPYf_dihedral_angle_d8.624713654

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