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- PDB-8ryt: Structural characterization of Thogoto Virus nucleoprotein provid... -

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Basic information

Entry
Database: PDB / ID: 8ryt
TitleStructural characterization of Thogoto Virus nucleoprotein provides insights into RNA encapsidation and assembly
ComponentsNucleoprotein
KeywordsVIRAL PROTEIN / Viral replication / nucleoprotein / RNA binding / oligomerization / orthomyxovirus
Function / homologyhelical viral capsid / viral penetration into host nucleus / host cell / viral nucleocapsid / ribonucleoprotein complex / symbiont entry into host cell / host cell nucleus / RNA binding / Nucleoprotein
Function and homology information
Biological speciesThogotovirus
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 18 Å
AuthorsRoske, Y. / Mikirtumov, V. / Daumke, O. / Kudryashev, M. / Dick, A.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)DA 1127/1-2 and SFB958/A12 Germany
CitationJournal: Structure / Year: 2024
Title: Structural characterization of Thogoto Virus nucleoprotein provides insights into viral RNA encapsidation and RNP assembly.
Authors: Alexej Dick / Vasilii Mikirtumov / Jonas Fuchs / Ferdinand Krupp / Daniel Olal / Elias Bendl / Thiemo Sprink / Christoph Diebolder / Mikhail Kudryashev / Georg Kochs / Yvette Roske / Oliver Daumke /
Abstract: Orthomyxoviruses, such as influenza and thogotoviruses, are important human and animal pathogens. Their segmented viral RNA genomes are wrapped by viral nucleoproteins (NPs) into helical ...Orthomyxoviruses, such as influenza and thogotoviruses, are important human and animal pathogens. Their segmented viral RNA genomes are wrapped by viral nucleoproteins (NPs) into helical ribonucleoprotein complexes (RNPs). NP structures of several influenza viruses have been reported. However, there are still contradictory models of how orthomyxovirus RNPs are assembled. Here, we characterize the crystal structure of Thogoto virus (THOV) NP and found striking similarities to structures of influenza viral NPs, including a two-lobed domain architecture, a positively charged RNA-binding cleft, and a tail loop important for trimerization and viral transcription. A low-resolution cryo-electron tomography reconstruction of THOV RNPs elucidates a left-handed double helical assembly. By providing a model for RNP assembly of THOV, our study suggests conserved NP assembly and RNA encapsidation modes for thogoto- and influenza viruses.
History
DepositionFeb 9, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Nucleoprotein
A: Nucleoprotein
B: Nucleoprotein
D: Nucleoprotein
E: Nucleoprotein
F: Nucleoprotein
G: Nucleoprotein
I: Nucleoprotein
K: Nucleoprotein
L: Nucleoprotein
M: Nucleoprotein
N: Nucleoprotein
O: Nucleoprotein
P: Nucleoprotein
Q: Nucleoprotein
R: Nucleoprotein


Theoretical massNumber of molelcules
Total (without water)814,13316
Polymers814,13316
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Nucleoprotein / Nucleocapsid protein / Protein N


Mass: 50883.324 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Details: Nucleoprotein / Source: (gene. exp.) Thogotovirus / Gene: Segment 5 / Cell line (production host): Fibroblasts / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P89216

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Ribonucleoprotein (RNP) complexes of Thogoto virus / Type: COMPLEX / Details: central part of RNP composed only NP molecules / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thogotovirus
Source (recombinant)Organism: Mesocricetus auratus (golden hamster)
Buffer solutionpH: 8
Details: 50 mM Tris (pH 8.0), 5 mM MgCl2, 100 mM KCl, 1.5 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 33000 X / Calibrated magnification: 33000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 3000 nm / Calibrated defocus min: 3000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 3.5 e/Å2 / Avg electron dose per subtomogram: 115.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 1
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1RELION4volume selection
2Dynamovolume selection
5GctfCTF correction
6RELION4CTF correction
9UCSF Chimeramodel fitting
10Cootmodel fitting
13RELION4final Euler assignment
14RELION4classification
15RELION43D reconstruction
16PHENIX1.20.1_4487:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -55.5 ° / Axial rise/subunit: 24.6 Å / Axial symmetry: C1
3D reconstructionResolution: 18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3128 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
EM volume selectionMethod: template matching with manual annotation on untilted images
Num. of tomograms: 33 / Num. of volumes extracted: 16101
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00353840
ELECTRON MICROSCOPYf_angle_d0.53872788
ELECTRON MICROSCOPYf_dihedral_angle_d4.2647172
ELECTRON MICROSCOPYf_chiral_restr0.0417734
ELECTRON MICROSCOPYf_plane_restr0.0049305

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