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- PDB-6n9y: Atomic structure of Non-Structural protein 1 of bluetongue virus -

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Basic information

Entry
Database: PDB / ID: 6n9y
TitleAtomic structure of Non-Structural protein 1 of bluetongue virus
ComponentsNon-structural protein 1
KeywordsVIRAL PROTEIN / Bluetongue Virus Non-structural protein 1
Function / homologyOrbivirus non-structural protein NS1/hydrophobic tubular protein / Orbivirus non-structural protein NS1, or hydrophobic tubular protein / Non-structural protein NS1
Function and homology information
Biological speciesBluetongue virus 23
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsKerviel, A. / Ge, P. / Lai, M. / Jih, J. / Boyce, M. / Zhang, X. / Zhou, Z.H. / Roy, P.
Funding support United States, United Kingdom, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
Wellcome Trust100218 United Kingdom
National Science Foundation (NSF, United States)MCB140140 United States
CitationJournal: Nat Microbiol / Year: 2019
Title: Atomic structure of the translation regulatory protein NS1 of bluetongue virus.
Authors: Adeline Kerviel / Peng Ge / Mason Lai / Jonathan Jih / Mark Boyce / Xing Zhang / Z Hong Zhou / Polly Roy /
Abstract: Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein ...Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells, but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an amino-terminal foot, a head and body domains connected to an extended carboxy-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc-finger-like motifs are present in each NS1 monomer, and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn, suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication, and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms.
History
DepositionDec 4, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1May 1, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 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 1.4Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Non-structural protein 1


Theoretical massNumber of molelcules
Total (without water)64,5551
Polymers64,5551
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area29620 Å2

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Components

#1: Protein Non-structural protein 1


Mass: 64554.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bluetongue virus 23 / References: UniProt: L7WU69

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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

ComponentName: Non-structural protein 1 (tubule) / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSaltNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Calibrated defocus min: 1200 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5006
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 50 / Used frames/image: 3-19

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN1.8particle selection
2Leginonimage acquisition
4CTFFIND3CTF correction
10RELION2final Euler assignment
11RELION1.4classificationIHRSR extension
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -17.5 ° / Axial rise/subunit: 8.83 Å / Axial symmetry: D2
3D reconstructionResolution: 4 Å / Resolution method: OTHER / Num. of particles: 7800 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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