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- EMDB-0142: Cryo-EM map of in vitro assembled Measles virus N into nucleocaps... -

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Entry
Database: EMDB / ID: EMD-0142
TitleCryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to viral genomic 5-prime RNA hexamers.
Map data
SampleCryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to viral genomic 5-prime RNA hexamers.:
Measles virus N / 5-prime RNA / NucleocapsidCapsid / nucleic-acidNucleic acid
Function / homologyParamyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / structural molecule activity / RNA binding / Nucleocapsid
Function and homology information
Biological speciesMeasles virus / synthetic construct (others) / Measles morbillivirus
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDesfosses A / Milles S / Ringkjobing Jensen M / Guseva S / Colletier JP / Maurin D / Schoehn G / Gutsche I / Ruigrok R / Blackledge M
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.
Authors: Ambroise Desfosses / Sigrid Milles / Malene Ringkjøbing Jensen / Serafima Guseva / Jacques-Philippe Colletier / Damien Maurin / Guy Schoehn / Irina Gutsche / Rob W H Ruigrok / Martin Blackledge /
Abstract: Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control ...Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.
Validation ReportPDB-ID: 6h5s

SummaryFull reportAbout validation report
DateDeposition: Jul 25, 2018 / Header (metadata) release: Aug 8, 2018 / Map release: Jun 12, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6h5s
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6h5s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0142.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 320 pix.
= 261.12 Å
0.82 Å/pix.
x 320 pix.
= 261.12 Å
0.82 Å/pix.
x 320 pix.
= 261.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.816 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.03520721 - 0.061960675
Average (Standard dev.)0.0004034026 (±0.003834821)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-160-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 261.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8160.8160.816
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z261.120261.120261.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-0.0350.0620.000

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Supplemental data

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Sample components

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Entire Cryo-EM map of in vitro assembled Measles virus N into nucleocaps...

EntireName: Cryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to viral genomic 5-prime RNA hexamers.
Number of components: 5

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Component #1: protein, Cryo-EM map of in vitro assembled Measles virus N into n...

ProteinName: Cryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to viral genomic 5-prime RNA hexamers.
Recombinant expression: No

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Component #2: protein, Measles virus N

ProteinName: Measles virus N / Recombinant expression: No
SourceSpecies: Measles virus
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, 5-prime RNA

ProteinName: 5-prime RNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, Nucleocapsid

ProteinName: NucleocapsidCapsid / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 46.425863 kDa
SourceSpecies: Measles morbillivirus
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: nucleic-acid, RNA (5'-R(*AP*CP*CP*AP*GP*A)-3')

nucleic acidName: RNA (5'-R(*AP*CP*CP*AP*GP*A)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
ACCAGA
MassTheoretical: 1.898229 kDa
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 3.934877693 Å / Delta phi: -29.15924186 %deg;
Sample solutionSpecimen conc.: 1 mg/mL
Buffer solution: 50 mM Na-phosphate pH 6, 150 mM NaCl, 2 mM DTT
pH: 6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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