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- PDB-9dou: Taeniopygia guttata R2 retrotransposon (R2Tg) initiating target-p... -

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Basic information

Entry
Database: PDB / ID: 9dou
TitleTaeniopygia guttata R2 retrotransposon (R2Tg) initiating target-primed reverse transcription
Components
  • (28S DNA bottom strand, ...) x 2
  • 28S DNA top strand
  • R2Tg 3'UTR RNA
  • R2Tg retrotransposon ORF
KeywordsRNA BINDING PROTEIN/RNA/DNA / retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTHYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTaeniopygia guttata (zebra finch)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWilkinson, M.E. / Edmonds, K.H.K. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2025
Title: Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons.
Authors: KeHuan K Edmonds / Max E Wilkinson / Daniel Strebinger / Hongyu Chen / Blake Lash / Clarissa C Schaefer / Shiyou Zhu / Dangliang Liu / Shai Zilberzwige-Tal / Alim Ladha / Michelle L Walsh / ...Authors: KeHuan K Edmonds / Max E Wilkinson / Daniel Strebinger / Hongyu Chen / Blake Lash / Clarissa C Schaefer / Shiyou Zhu / Dangliang Liu / Shai Zilberzwige-Tal / Alim Ladha / Michelle L Walsh / Chris J Frangieh / Nicholas A Vaz Reay / Rhiannon K Macrae / Xiao Wang / Feng Zhang /
Abstract: R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our ...R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3' UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system's size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5' end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools.
History
DepositionSep 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: R2Tg retrotransposon ORF
B: 28S DNA bottom strand, 3' side
P: 28S DNA bottom strand, 5' side (priming strand)
R: R2Tg 3'UTR RNA
T: 28S DNA top strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)398,92811
Polymers398,1595
Non-polymers7686
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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28S DNA bottom strand, ... , 2 types, 2 molecules BP

#2: DNA chain 28S DNA bottom strand, 3' side


Mass: 65051.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)
#3: DNA chain 28S DNA bottom strand, 5' side (priming strand)


Mass: 3997.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)

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Protein / RNA chain / DNA chain , 3 types, 3 molecules ART

#1: Protein R2Tg retrotransposon ORF


Mass: 157228.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Taeniopygia guttata (zebra finch) / Production host: Saccharomyces cerevisiae (brewer's yeast)
#4: RNA chain R2Tg 3'UTR RNA


Mass: 106656.273 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)
#5: DNA chain 28S DNA top strand


Mass: 65225.641 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Taeniopygia guttata (zebra finch)

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Non-polymers , 3 types, 6 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationN

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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: Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.9
Details: 20 mM HEPES-KOH pH 7.9, 400 mM potassium acetate, 5 mM magnesium acetate, 0.1 mM dTTP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: A260 = 17
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 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: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.02 sec. / Electron dose: 59.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10627
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
12RELION5final Euler assignment
13RELION5classification
14RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 323700
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30612 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611015
ELECTRON MICROSCOPYf_angle_d1.0915539
ELECTRON MICROSCOPYf_dihedral_angle_d23.4212831
ELECTRON MICROSCOPYf_chiral_restr0.0571785
ELECTRON MICROSCOPYf_plane_restr0.0091447

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