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- EMDB-0141: Cryo-EM structure of in vitro assembled Measles virus N into nucl... -

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Basic information

Entry
Database: EMDB / ID: EMD-0141
TitleCryo-EM structure of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
Map dataCryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
Sample
  • Complex: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
    • Complex: Measles virus Nucleoprotein
      • Protein or peptide: NucleocapsidCapsid
    • Complex: polyA RNA
      • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
Function / homologyParamyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / 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 J / Maurin D / Schoehn G / Gutsche I / Ruigrok R / Blackledge M
Funding support France, 5 items
OrganizationGrant numberCountry
European Molecular Biology OrganizationALTF 468-2014 France
FRISBIANR-10-INSB-05-02 France
GRALANR-10-LABX-49-01 France
Fondation Recherche Medicale (FRM)ARF20160936266 France
European CommissionEMBOCOFUND2012, GA-2012-600394
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.
History
DepositionJul 25, 2018-
Header (metadata) releaseAug 8, 2018-
Map releaseMar 13, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

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  • 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
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  • Surface view with fitted model
  • Atomic models: PDB-6h5q
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6h5q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0141.png

Downloads & links

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Map

FileDownload / File: emd_0141.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
Voxel sizeX=Y=Z: 0.816 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.0512 - 0.08295685
Average (Standard dev.)0.00043131955 (±0.0054542553)
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-0.0510.0830.000

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Supplemental data

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Sample components

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Entire : Measles virus Nucleoprotein assembled into nucleocapsid-like part...

EntireName: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
Components
  • Complex: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
    • Complex: Measles virus Nucleoprotein
      • Protein or peptide: NucleocapsidCapsid
    • Complex: polyA RNA
      • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')

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Supramolecule #1: Measles virus Nucleoprotein assembled into nucleocapsid-like part...

SupramoleculeName: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Measles virus Nucleoprotein

SupramoleculeName: Measles virus Nucleoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Measles virus
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: polyA RNA

SupramoleculeName: polyA RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Nucleocapsid

MacromoleculeName: Nucleocapsid / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Measles morbillivirus
Molecular weightTheoretical: 46.425863 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MATLLRSLAL FKRNKDKPPI TSGSGGAIRG IKHIIIVPIP GDSSITTRSR LLDRLVRLIG NPDVSGPKLT GALIGILSLF VESPGQLIQ RITDDPDVSI RLLEVVQSDQ SQSGLTFASR GTNMEDEADQ YFSHDDPISS DQSRFGWFEN KEISDIEVQD P EGFNMILG ...String:
MATLLRSLAL FKRNKDKPPI TSGSGGAIRG IKHIIIVPIP GDSSITTRSR LLDRLVRLIG NPDVSGPKLT GALIGILSLF VESPGQLIQ RITDDPDVSI RLLEVVQSDQ SQSGLTFASR GTNMEDEADQ YFSHDDPISS DQSRFGWFEN KEISDIEVQD P EGFNMILG TILAQIWVLL AKAVTAPDTA ADSELRRWIK YTQQRRVVGE FRLERKWLDV VRNRIAEDLS LRRFMVALIL DI KRTPGNK PRIAEMICDI DTYIVEAGLA SFILTIKFGI ETMYPALGLH EFAGELSTLE SLMNLYQQMG ETAPYMVILE NSI QNKFSA GSYPLLWSYA MGVGVELENS MGGLNFGRSY FDPAYFRLGQ EMVRRSAGKV SSTLASELGI TAEDARLVSE IAMH TTEDK VEHHHHHHHH

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Macromolecule #2: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')

MacromoleculeName: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.930277 KDa
SequenceString:
AAAAAA

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 6 / Details: 50 mM Na-phosphate pH 6, 150 mM NaCl, 2 mM DTT
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.0008 µm / Nominal magnification: 23000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 186 / Average exposure time: 4.0 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 111582 / Software - Name: RELION (ver. 2.1)
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

2867

Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Helical parameters - Δz: 3.920265781 Å
Applied symmetry - Helical parameters - Δ&Phi: -29.17105583 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 111582

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