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- PDB-8a5t: Capsid structure of the L-A helper virus from native viral communities -

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Basic information

Entry
Database: PDB / ID: 8a5t
TitleCapsid structure of the L-A helper virus from native viral communities
ComponentsMajor capsid protein
KeywordsVIRUS / Capsid structure ScVLA / viral particle / wildtype / endogenous
Function / homologyMajor coat protein, L-A virus / L-A virus major coat protein superfamily / L-A virus, major coat protein / viral capsid / viral translational frameshifting / Major capsid protein
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsSchmidt, L. / Tueting, C. / Stubbs, M.T. / Kastritis, P.L.
Funding support Germany, European Union, 5items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research03Z22HN23 Germany
German Federal Ministry for Education and Research03Z22HI2 Germany
German Federal Ministry for Education and Research03COV04 Germany
German Research Foundation (DFG)391498659 Germany
European Regional Development FundEFRE: ZS/2016/04/78115European Union
Citation
Journal: Commun Biol / Year: 2024
Title: Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.
Authors: Lisa Schmidt / Christian Tüting / Fotis L Kyrilis / Farzad Hamdi / Dmitry A Semchonok / Gerd Hause / Annette Meister / Christian Ihling / Milton T Stubbs / Andrea Sinz / Panagiotis L Kastritis /
Abstract: The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to ...The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.
#1: Journal: Commun Biol / Year: 2024
Title: Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.
Authors: Lisa Schmidt / Christian Tüting / Fotis L Kyrilis / Farzad Hamdi / Dmitry A Semchonok / Gerd Hause / Annette Meister / Christian Ihling / Milton T Stubbs / Andrea Sinz / Panagiotis L Kastritis /
Abstract: The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to ...The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.
#2: Journal: Biorxiv / Year: 2022
Title: Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts
Authors: Schmidt, L. / Tuting, C. / Kyrilis, F.L. / Hamdi, F. / Semchonok, D.A. / Hause, G. / Meister, A. / Ihling, C. / Shah, P.N.M. / Stubbs, M.T. / Sinz, A. / Stuart, D.I. / Kastritis, P.L.
History
DepositionJun 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Major capsid protein
B: Major capsid protein


Theoretical massNumber of molelcules
Total (without water)152,1402
Polymers152,1402
Non-polymers00
Water00
1
A: Major capsid protein
B: Major capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)9,128,404120
Polymers9,128,404120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Major capsid protein / Gag protein / Major coat protein


Mass: 76070.031 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32503

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Saccharomyces cerevisiae virus L-A / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae virus L-A
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Saccharomyces cerevisiae
Buffer solutionpH: 7.4
Details: pH of the buffer was adjusted with NaOH buffer was filtered and sonicated
Buffer componentConc.: 200 mM / Name: Ammoniumacetate / Formula: CH3COONH4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: heterogenous cell extract
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K / Details: blot force 2 and blot time 6 s before plunging

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Calibrated magnification: 89297 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 118 K / Temperature (min): 77 K
Image recordingAverage exposure time: 3.61 sec. / Electron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7020
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3particle selection
2EPU2.11.1.11RELimage acquisition
4cryoSPARCv.3.3CTF correction
7UCSF ChimeraX1.3model fittingInitial model was ridig-fitted into the density
9Coot0.9.6model refinementManual refinement of the model
10PHENIXdev-4379model refinementreal_space_refinement with default parameters
13cryoSPARCv3.3classification
14cryoSPARCv3.33D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2725140
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1020420 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: The initial model was rigid-fitted by ChimeraX and refined by iterative cycles of Coot and PHENIX.
Atomic model buildingPDB-ID: 1M1C

1m1c


Accession code: 1M1C / Source name: PDB / Type: experimental model

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