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- EMDB-6337: Structure of the L-protein of vesicular stomatitis virus from ele... -

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Basic information

Entry
Database: EMDB / ID: EMD-6337
TitleStructure of the L-protein of vesicular stomatitis virus from electron cryomicroscopy
Map dataReconstruction of the L-protein of vesicular stomatitis virus
Sample
  • Sample: VSV-L
  • Protein or peptide: VSV large protein
  • Protein or peptide: VSV phosphoprotein
KeywordsRNA-dependent RNA polymerase / RNA capping / cryoEM single-particle analysis
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / viral transcription / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity ...NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / viral transcription / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding
Similarity search - Function
: / Virus-capping methyltransferase, C-terminal / : / Virus-capping methyltransferase, connector domain / 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 ...: / Virus-capping methyltransferase, C-terminal / : / Virus-capping methyltransferase, connector domain / 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
RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesVesicular stomatitis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLiang B / Li Z / Jenni S / Rameh AA / Morin BM / Grant T / Grigorieff N / Harrison SC / Whelan SPJ
CitationJournal: Cell / Year: 2015
Title: Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy.
Authors: Bo Liang / Zongli Li / Simon Jenni / Amal A Rahmeh / Benjamin M Morin / Timothy Grant / Nikolaus Grigorieff / Stephen C Harrison / Sean P J Whelan /
Abstract: The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as ...The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.
History
DepositionMay 7, 2015-
Header (metadata) releaseMay 20, 2015-
Map releaseMay 20, 2015-
UpdateAug 19, 2015-
Current statusAug 19, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a22
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6337.gif

Downloads & links

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Map

FileDownload / File: emd_6337.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the L-protein of vesicular stomatitis virus
Voxel sizeX=Y=Z: 1.237 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-2.92821145 - 5.04094791
Average (Standard dev.)0.05180021 (±0.38479805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 148.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2371.2371.237
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z148.440148.440148.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-2.9285.0410.052

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Supplemental data

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Sample components

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Entire : VSV-L

EntireName: VSV-L
Components
  • Sample: VSV-L
  • Protein or peptide: VSV large protein
  • Protein or peptide: VSV phosphoprotein

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Supramolecule #1000: VSV-L

SupramoleculeName: VSV-L / type: sample / ID: 1000
Oligomeric state: One monomer of VSV-L bound to one monomer of P
Number unique components: 2
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: VSV large protein

MacromoleculeName: VSV large protein / type: protein_or_peptide / ID: 1 / Name.synonym: VSV-L / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Vesicular stomatitis virus
Molecular weightTheoretical: 240 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf21

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Macromolecule #2: VSV phosphoprotein

MacromoleculeName: VSV phosphoprotein / type: protein_or_peptide / ID: 2 / Name.synonym: P / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Vesicular stomatitis virus
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.4 / Details: 25 mM HEPES, 250 mM NaCl, 6 mM MgSO4, 0.5 mM TCEP
GridDetails: 400 mesh Quantifoil R1.2/1.3 Cu grid
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Instrument: FEI VITROBOT MARK I
Method: Blot time 2 seconds, drain time 1 second before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40410 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.3 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 29000
Sample stageSpecimen holder: CT3500 / Specimen holder model: GATAN LIQUID NITROGEN
DetailsBeam intensity: 8 electrons/pixel/s Movie mode: 30 frames, 5 frames/s
DateAug 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 1272 / Average electron dose: 31 e/Å2
Details: Images are the sums of all 30 aligned movie frames (high dose) or frames 3 - 12 (low-dose).
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 3
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: EMAN2, IMAGIC, Frealign
Details: The final map represents the best class out of three classes.
Number images used: 74940
DetailsAn initial map was obtained with EMAN2, IMAGIC, and TIGRIS. CTF was determined using CTFFIND3. Refinement and classification were done using Frealign.

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