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- PDB-6r24: The structure of a Ty3 retrotransposon icosahedral capsid -

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Basic information

Entry
Database: PDB / ID: 6r24
TitleThe structure of a Ty3 retrotransposon icosahedral capsid
ComponentsTransposon Ty3-I Gag-Pol polyprotein
KeywordsVIRUS LIKE PARTICLE / Ty3 / retrotransposon / retrovirus / capsid / Gag polyprotein
Function / homology
Function and homology information


retrotransposon nucleocapsid / retrotransposition / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / RNA nuclease activity / DNA integration / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / peptidase activity ...retrotransposon nucleocapsid / retrotransposition / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / RNA nuclease activity / DNA integration / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / peptidase activity / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / proteolysis / DNA binding / RNA binding / zinc ion binding / ATP binding / nucleus / cytoplasm
Similarity search - Function
Peptidase A2B, Ty3 transposon peptidase / Ty3 transposon peptidase / Ty3 transposon capsid-like protein / Ty3 transposon capsid-like protein / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / Integrase core domain / Integrase, catalytic core ...Peptidase A2B, Ty3 transposon peptidase / Ty3 transposon peptidase / Ty3 transposon capsid-like protein / Ty3 transposon capsid-like protein / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / zinc finger / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Transposon Ty3-I Gag-Pol polyprotein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsDodonova, S.O. / Prinz, S. / Bilanchone, V. / Sandmeyer, S. / Briggs, J.A.G.
Funding support Germany, United Kingdom, 2items
OrganizationGrant numberCountry
German Research FoundationBR 3635/2-1 Germany
Medical Research Council (United Kingdom)MC_UP_1201/16 United 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.
History
DepositionMar 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2019Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria

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Assembly

Deposited unit
A: Transposon Ty3-I Gag-Pol polyprotein
B: Transposon Ty3-I Gag-Pol polyprotein
C: Transposon Ty3-I Gag-Pol polyprotein
D: Transposon Ty3-I Gag-Pol polyprotein
E: Transposon Ty3-I Gag-Pol polyprotein
F: Transposon Ty3-I Gag-Pol polyprotein
G: Transposon Ty3-I Gag-Pol polyprotein
H: Transposon Ty3-I Gag-Pol polyprotein
I: Transposon Ty3-I Gag-Pol polyprotein


Theoretical massNumber of molelcules
Total (without water)327,4419
Polymers327,4419
Non-polymers00
Water0
1
A: Transposon Ty3-I Gag-Pol polyprotein
B: Transposon Ty3-I Gag-Pol polyprotein
C: Transposon Ty3-I Gag-Pol polyprotein
D: Transposon Ty3-I Gag-Pol polyprotein
E: Transposon Ty3-I Gag-Pol polyprotein
F: Transposon Ty3-I Gag-Pol polyprotein
G: Transposon Ty3-I Gag-Pol polyprotein
H: Transposon Ty3-I Gag-Pol polyprotein
I: Transposon Ty3-I Gag-Pol polyprotein
x 60


Theoretical massNumber of molelcules
Total (without water)19,646,480540
Polymers19,646,480540
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation59
MethodUCSF CHIMERA

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Components

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


Mass: 36382.371 Da / Num. of mol.: 9 / Mutation: D336I
Source method: isolated from a genetically manipulated source
Details: D336I mutation in the active center of the Ty3 protease
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 (brewer's yeast)
Strain (production host): 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: T9 icosahedral capsid of a Ty3 retrotransposon / Type: COMPLEX
Details: The asymmetric unit contains 9 monomers of the Ty3 capsid molecule.
Entity ID: all / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: yVB1680 strain - BY4741 killer minus, Ty3 null / Plasmid: pJK776
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
IDConc.NameFormulaBuffer-ID
110 mMTris1
2100 mMNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid1
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% ...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 ...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 µm
Specimen holderCryogen: NITROGEN / Specimen holder 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
7UCSF Chimera1.13model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1727
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1236 / 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: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient

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