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- EMDB-2930: Cryo-electron microscopy map of La Crosse orthobunyavirus polymer... -

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Basic information

Entry
Database: EMDB / ID: EMD-2930
TitleCryo-electron microscopy map of La Crosse orthobunyavirus polymerase (apo form, residues 1-1750)
Map dataCryo-electron microscopy map of La Crosse orthobunyavirus polymerase (apo form, residues 1-1750)
Sample
  • Sample: La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)
  • Protein or peptide: La Crosse RNA-dependent RNA polymerase
KeywordsRNA dependent RNA polymerase / segmented negative stranded RNA virus / replication / transcription
Function / homology
Function and homology information


host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding ...host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, orthobunyavirus / : / Virus, RNA-directed RNA polymerase L, thumb ring domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / : / RNA-dependent RNA polymerase, bunyaviral / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesLa Crosse virus
Methodsingle particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsGerlach P / Malet H / Cusack S / Reguera J
CitationJournal: Cell / Year: 2015
Title: Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter.
Authors: Piotr Gerlach / Hélène Malet / Stephen Cusack / Juan Reguera /
Abstract: Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, ...Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3' and 5' vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5' end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.
History
DepositionMar 12, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseJun 10, 2015-
UpdateJun 17, 2015-
Current statusJun 17, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.148
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.148
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image_EMD-29...

Downloads & links

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Map

FileDownload / File: emd_2930.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron microscopy map of La Crosse orthobunyavirus polymerase (apo form, residues 1-1750)
Voxel sizeX=Y=Z: 2.02 Å
Density
Contour LevelBy AUTHOR: 0.148 / Movie #1: 0.148
Minimum - Maximum-0.5006029 - 0.65991658
Average (Standard dev.)0.00293544 (±0.02737703)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 242.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.022.022.02
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z242.400242.400242.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.5010.6600.003

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Supplemental data

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Sample components

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Entire : La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)

EntireName: La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)
Components
  • Sample: La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)
  • Protein or peptide: La Crosse RNA-dependent RNA polymerase

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Supramolecule #1000: La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)

SupramoleculeName: La Crosse orthobunyavirus polymerase (residues 1-1750, apo form)
type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 203.5 KDa / Theoretical: 203.5 KDa

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Macromolecule #1: La Crosse RNA-dependent RNA polymerase

MacromoleculeName: La Crosse RNA-dependent RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: La Crosse virus / Strain: LACV/mosquito/1978 / Location in cell: cytoplasm
Molecular weightExperimental: 20.35 KDa / Theoretical: 20.35 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: Hi5 / Recombinant plasmid: pFastBac
SequenceUniProtKB: RNA-directed RNA polymerase L / GO: RNA-dependent RNA polymerase activity / InterPro: RNA-dependent RNA polymerase, bunyaviral

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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

Concentration0.2 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCl, 150 mM NaCl, 2 mM TCEP, pH 8
GridDetails: Quantifoil grid 400 mesh 2/1
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 2s before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 80 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 138129 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 138129
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 100 K
DetailsBinning factor 2 used for data collection
DateMar 11, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 28 µm / Number real images: 6129 / Average electron dose: 14 e/Å2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: OTHER / Software - Name: RELION1.3 / Number images used: 178983
DetailsCTF correction using CTFFIND3 Manual picking in EMAN boxer 2D classification in IMAGIC5 and RELION1.3 Automated picking using the fast projection matching algorithm Refinement and 3D classification in RELION1.3

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Atomic model buiding 1

Initial modelPDB ID:

5amr


Chain - Chain ID: A
SoftwareName: Chimera
DetailsTwo rigid bodies were used: 1-184 185-1745
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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