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- PDB-6r23: The structure of a Ty3 retrotransposon capsid C-terminal domain dimer -

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Entry
Database: PDB / ID: 6r23
TitleThe structure of a Ty3 retrotransposon capsid C-terminal domain dimer
ComponentsTransposon Ty3-I Gag-Pol polyprotein
KeywordsVIRUS LIKE PARTICLE / Ty3 / retrotransposon / retrovirus / capsid / Gag polyprotein / capsid-CTD
Function / homology
Function and homology information


retrotransposon nucleocapsid / transposition, RNA-mediated / Hydrolases, Acting on peptide bonds (peptidases), Aspartic endopeptidases / ribonuclease H / ribonuclease activity / RNA-directed DNA polymerase / DNA integration / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / peptidase activity ...retrotransposon nucleocapsid / transposition, RNA-mediated / Hydrolases, Acting on peptide bonds (peptidases), Aspartic endopeptidases / ribonuclease H / ribonuclease activity / RNA-directed DNA polymerase / DNA integration / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / peptidase activity / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / aspartic-type endopeptidase activity / RNA binding / DNA binding / zinc ion binding / ATP binding / nucleus / cytoplasm
Integrase core domain / Ribonuclease H-like superfamily / Integrase, catalytic core / Zinc finger, CCHC-type / Ty3 transposon peptidase / Zinc finger CCHC-type profile. / Reverse transcriptase (RT) catalytic domain profile. / Integrase catalytic domain profile. / Aspartic peptidase domain superfamily / Peptidase A2B, Ty3 transposon peptidase ...Integrase core domain / Ribonuclease H-like superfamily / Integrase, catalytic core / Zinc finger, CCHC-type / Ty3 transposon peptidase / Zinc finger CCHC-type profile. / Reverse transcriptase (RT) catalytic domain profile. / Integrase catalytic domain profile. / Aspartic peptidase domain superfamily / Peptidase A2B, Ty3 transposon peptidase / Ribonuclease H superfamily / Zinc finger, CCHC-type superfamily / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain
Transposon Ty3-I Gag-Pol polyprotein
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsDodonova, S.O. / Prinz, S. / Bilanchone, V. / Sandmeyer, S. / Briggs, J.A.G.
Funding supportGermany , United Kingdom , 2件
OrganizationGrant numberCountry
German Research FoundationBR 3635/2-1Germany
Medical Research Council (United Kingdom)MC_UP_1201/16United Kingdom
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses.
Authors: Svetlana O Dodonova / Simone Prinz / Virginia Bilanchone / Suzanne Sandmeyer / John A G Briggs /
Abstract: Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR ...Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation. They encode a Gag polypeptide, the capsid domains of which can oligomerize to form a virus-like particle. The structures of retroviral capsids have been extensively described. They assemble an immature viral particle through oligomerization of full-length Gag. Proteolytic cleavage of Gag results in a mature, infectious particle. In contrast, the absence of structural data on LTR retrotransposon capsids hinders our understanding of their function and evolutionary relationships. Here, we report the capsid morphology and structure of the archetypal Gypsy retrotransposon Ty3. We performed electron tomography (ET) of immature and mature Ty3 particles within cells. We found that, in contrast to retroviruses, these do not change size or shape upon maturation. Cryo-ET and cryo-electron microscopy of purified, immature Ty3 particles revealed an irregular fullerene geometry previously described for mature retrovirus core particles and a tertiary and quaternary arrangement of the capsid (CA) C-terminal domain within the assembled capsid that is conserved with mature HIV-1. These findings provide a structural basis for studying retrotransposon capsids, including those domesticated in higher organisms. They suggest that assembly via a structurally distinct immature capsid is a later retroviral adaptation, while the structure of mature assembled capsids is conserved between LTR retrotransposons and retroviruses.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 15, 2019 / Release: May 8, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 8, 2019Structure modelrepositoryInitial release
1.1May 22, 2019Structure modelData collection / Database referencescitation / em_admin / pdbx_database_proc_citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Transposon Ty3-I Gag-Pol polyprotein
B: Transposon Ty3-I Gag-Pol polyprotein


Theoretical massNumber of molelcules
Total (without water)72,7652
Polymers72,7652
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1050 Å2
ΔGint-7 kcal/mol
Surface area13580 Å2
MethodPISA

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Components

#1: Protein/peptide Transposon Ty3-I Gag-Pol polyprotein / Gag3-Pol3 / Transposon Ty3-2 TYA-TYB polyprotein


Mass: 36382.371 Da / Num. of mol.: 2
Details: D336I mutation in the active center of the Ty3 protease
Mutation: D336I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: TY3B-I, YILWTy3-1 POL, YIL082W-A / Plasmid: pJK776 / Production host: Saccharomyces cerevisiae (baker's yeast)
Strain (production host): strain yVB1680 strain - BY4741 killer minus, Ty3 null
References: UniProt: Q7LHG5, Hydrolases, Acting on peptide bonds (peptidases), Aspartic endopeptidases, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H

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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: Ty3 capsid C-terminal domain dimer / Type: COMPLEX
Details: The 9 non-symmetry related copies of the Ty3 Capsid-CTD from the complete capsid shell structure were aligned and averaged to generate a higher resolution 4.9 A structure
Entity ID: 1 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Saccharomyces cerevisiae
Virus shellName: GAG Capsid / Diameter: 480 nm / Triangulation number (T number): 9
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.NameFormula
110 mMTris
2100 mMNaCl
31 mMEDTAEthylenediaminetetraacetic acid
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 296 K
Details: The sample was applied onto glow-discharged C-flat (Protochips Inc.) holey carbon grids. The grids were blotted from the back side for 11 seconds at room temperature in a chamber at 85% humidity and plunge-frozen into liquid ethane using a manual plunger.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Cryo-grids containing purified PR- Ty3 particles were imaged in a Titan Krios electron microscope equipped with a Falcon II direct electron detector, operated at 300 kV. Images were collected with a nominal magnification of 75000, giving a pixel size of 1.08 A. Images were collected in integrating mode with a total electron dose of 20 e/A2. The range of applied defocus values was between -1.0 um and -3.5 um.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µns
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1
Image scansMovie frames/image: 1

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7MDFFmodel fitting
9PHENIX1.14model refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13TOM Toolbox3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1727
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74160
Details: The Ty3 particles have a T=9 and each asymmetric unit of the lattice contains nine non-symmetry related Ty3 capsid protein monomers. We extracted all non-symmetry related units from the final reconstructions from the two half datasets. The CA-CTDs were aligned and averaged separately. The final structure of the CA-CTD comes from nine averaged copies of the domain. The final structure of the CA-CTD was resolved at 4.9 A resolution at 0.143 FSC. Gaussian-smoothened ellipsoid-shaped masks were used for resolution measurements.
Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionDetails: 1733 vesicles and near-complete buds were picked from 61 tomograms. Subtomograms were extracted from the surface of the vesicles.
Num. of tomograms: 54 / Num. of volumes extracted: 2547
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

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