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- PDB-9nl3: Structure of R2 retrotransposon protein from Taeniopygia guttata ... -

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Basic information

Entry
Database: PDB / ID: 9nl3
TitleStructure of R2 retrotransposon protein from Taeniopygia guttata initiating target-primed reverse transcription
Components
  • 3'UTR RNA
  • Bottom strand for target rDNA
  • Primer
  • R2 retrotransposon protein
  • Top strand for target rDNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / Retrotransposon / Reverse transcriptase / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTHYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTaeniopygia guttata (zebra finch)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsThawani, A. / Collins, K. / Nogales, E.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP1 HL156819 United States
CitationJournal: Sci Adv / Year: 2025
Title: Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second-strand nicking.
Authors: Akanksha Thawani / Anthony Rodríguez-Vargas / Briana Van Treeck / Nozhat T Hassan / David L Adelson / Eva Nogales / Kathleen Collins /
Abstract: R2 retrotransposons are site-specific eukaryotic non-long terminal repeat retrotransposons that copy and paste into gene loci encoding ribosomal RNAs. Recently, we demonstrated that avian A-clade R2 ...R2 retrotransposons are site-specific eukaryotic non-long terminal repeat retrotransposons that copy and paste into gene loci encoding ribosomal RNAs. Recently, we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not well characterized. Here, we report high-resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins at the initiation of target-primed reverse transcription and after cDNA synthesis and second-strand nicking. Using biochemical and cellular assays, we illuminate the basis for high selectivity of template use and unique roles for each of the three zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif specific to vertebrate A-clade R2 proteins. Our work provides the first insights into A-clade R2 protein structure during gene insertion and may enable future improvement and adaptation of R2-based systems for precise transgene insertion.
History
DepositionMar 2, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 18, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 2, 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 / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: R2 retrotransposon protein
B: Bottom strand for target rDNA
P: Primer
R: 3'UTR RNA
T: Top strand for target rDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)277,01211
Polymers276,2445
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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DNA chain , 3 types, 3 molecules BPT

#2: DNA chain Bottom strand for target rDNA


Mass: 21507.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Primer


Mass: 3997.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain Top strand for target rDNA


Mass: 21654.875 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AR

#1: Protein R2 retrotransposon protein


Mass: 133494.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Taeniopygia guttata (zebra finch) / Production host: Escherichia coli (E. coli)
#4: RNA chain 3'UTR RNA


Mass: 95589.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 6 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3

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Details

Has ligand of interestY
Has protein modificationY

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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: R2 retrotransposon protein in TPRT initiation stage / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5096
Image scansWidth: 11520 / Height: 8184

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18892 / Symmetry type: POINT

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