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- PDB-6h5q: Cryo-EM structure of in vitro assembled Measles virus N into nucl... -

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Basic information

Entry
Database: PDB / ID: 6h5q
TitleCryo-EM structure of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.
Components
  • Nucleocapsid
  • RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
KeywordsVIRAL PROTEIN / Measles / Nucleocapsid / Helical / RNA
Function / homology
Function and homology information


helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / host cell nucleus / structural molecule activity / RNA binding
Similarity search - Function
Paramyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein
Similarity search - Domain/homology
Biological speciesMeasles morbillivirus
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical 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, 5items
OrganizationGrant numberCountry
European Molecular Biology OrganizationALTF 468-2014 France
GRALANR-10-LABX-49-01 France
FRISBIANR-10-INSB-05-02 France
European CommissionEMBOCOFUND2012, GA-2012-600394
Fondation Recherche Medicale (FRM)ARF20160936266 France
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, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Refinement description / Category: refine / Item: _refine.pdbx_refine_id
Revision 1.2Nov 6, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Assembly

Deposited unit
B: Nucleocapsid
R: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)48,3562
Polymers48,3562
Non-polymers00
Water00
1
B: Nucleocapsid
R: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
x 15


Theoretical massNumber of molelcules
Total (without water)725,34230
Polymers725,34230
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation14
MethodUCSF CHIMERA

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Components

#1: Protein Nucleocapsid / Nucleocapsid protein


Mass: 46425.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Measles morbillivirus / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: B8PZP0
#2: RNA chain RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')


Mass: 1930.277 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers.COMPLEXall0MULTIPLE SOURCES
2Measles virus NucleoproteinCOMPLEX#11RECOMBINANT
3polyA RNACOMPLEX#21RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Measles virus11234
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 6 / Details: 50 mM Na-phosphate pH 6, 150 mM NaCl, 2 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 23000 X / Nominal defocus max: 3 nm / Nominal defocus min: 0.8 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 4 sec. / Electron dose: 30 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 186
Image scansSampling size: 5 µm / Width: 4000 / Height: 4000 / Movie frames/image: 40 / Used frames/image: 2-20

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2LatitudeSimage acquisition
4GctfCTF correction
9SPRINGinitial Euler assignment
10RELION2.1final Euler assignment
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -29.17105583 ° / Axial rise/subunit: 3.920265781 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 111582
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111582 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
RefinementHighest resolution: 3.3 Å

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