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- PDB-7oi3: Cryo-EM structure of the Cetacean morbillivirus nucleoprotein-RNA... -

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Basic information

Entry
Database: PDB / ID: 7oi3
TitleCryo-EM structure of the Cetacean morbillivirus nucleoprotein-RNA complex
Components
  • Cetacean morbillivirus nucleoprotein
  • poly-A 6-mer
KeywordsVIRAL PROTEIN / Cetacean morbillivirus / nucleocapsid / RNA virus replication / cryo-electron microscopy / helical reconstruction / single particle analysis
Function / homologyParamyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / structural molecule activity / RNA binding / RNA / Nucleocapsid
Function and homology information
Biological speciesMorbillivirus sp.
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsZinzula, L. / Beck, F. / Klumpe, S. / Bohn, S. / Pfeifer, G. / Bollschweiler, D. / Nagy, I. / Plitzko, J.M. / Baumeister, W.
CitationJournal: J Struct Biol / Year: 2021
Title: Cryo-EM structure of the cetacean morbillivirus nucleoprotein-RNA complex.
Authors: Luca Zinzula / Florian Beck / Sven Klumpe / Stefan Bohn / Günter Pfeifer / Daniel Bollschweiler / István Nagy / Jürgen M Plitzko / Wolfgang Baumeister /
Abstract: Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and ...Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and conservation of endangered marine mammal populations in both hemispheres. As for all non-segmented, negative-sense, single-stranded RNA (ssRNA) viruses, the morbilliviral genome is enwrapped by thousands of nucleoprotein (N) protomers. Each bound to six ribonucleotides, N protomers assemble to form a helical ribonucleoprotein (RNP) complex that serves as scaffold for nucleocapsid formation and as template for viral replication and transcription. While the molecular details on RNP complexes elucidated in human measles virus (MeV) served as paradigm model for these processes in all members of the Morbillivirus genus, no structural information has been obtained from other morbilliviruses, nor has any CeMV structure been solved so far. We report the structure of the CeMV RNP complex, reconstituted in vitro upon binding of recombinant CeMV N to poly-adenine ssRNA hexamers and solved to 4.0 Å resolution by cryo-electron microscopy. In spite of the amino acid sequence similarity and consequently similar folding of the N protomer, the CeMV RNP complex exhibits different helical parameters as compared to previously reported MeV orthologs. The CeMV structure reveals exclusive interactions leading to more extensive protomer-RNA and protomer-protomer interfaces. We identified twelve residues, among those varying between CeMV strains, as putatively important for the stabilization of the RNP complex, which highlights the need to study the potential of CeMV N mutations that modulate nucleocapsid assembly to also affect viral phenotype and host adaptation.
History
DepositionMay 11, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 23, 2021Provider: repository / Type: Initial release

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Structure viewerMolecule:
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Assembly

Deposited unit
E: Cetacean morbillivirus nucleoprotein
B: poly-A 6-mer


Theoretical massNumber of molelcules
Total (without water)48,2042
Polymers48,2042
Non-polymers00
Water0
1
E: Cetacean morbillivirus nucleoprotein
B: poly-A 6-mer
x 15


Theoretical massNumber of molelcules
Total (without water)723,05930
Polymers723,05930
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation14

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Components

#1: Protein Cetacean morbillivirus nucleoprotein


Mass: 46273.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Morbillivirus sp. / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1I9RYK9
#2: RNA chain poly-A 6-mer


Mass: 1930.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Morbillivirus sp. / Production host: 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
1Cetacean morbillivirus nucleoprotein-RNA complexCOMPLEXall0RECOMBINANT
2Cetacean morbillivirus nucleoproteinCOMPLEX#11RECOMBINANT
3poly-A 6-merCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Morbillivirus sp.2078987
33Morbillivirus sp.2078987
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Synthetic construct (others)32630
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaClSodium chloride1
220 mMTris1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3153

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
3Latitudeimage acquisition
5CTFFINDCTF correction
11RELIONinitial Euler assignment
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 28.5 ° / Axial rise/subunit: 5 Å / Axial symmetry: C1
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31361 / Symmetry type: HELICAL

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