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Yorodumi- EMDB-47091: Taeniopygia guttata R2 retrotransposon (R2Tg) initiating target-p... -
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Basic information
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| Title | Taeniopygia guttata R2 retrotransposon (R2Tg) initiating target-primed reverse transcription | |||||||||
Map data | Sharpened map of Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription | |||||||||
Sample |
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Keywords | retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
| Biological species | ![]() | |||||||||
| Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Wilkinson ME / Edmonds KHK / Zhang F | |||||||||
| Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2025Title: 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. | |||||||||
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Structure visualization
| Supplemental images |
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Downloads & links
-EMDB archive
| Map data | emd_47091.map.gz | 84.8 MB | EMDB map data format | |
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| Header (meta data) | emd-47091-v30.xml emd-47091.xml | 27.8 KB 27.8 KB | Display Display | EMDB header |
| FSC (resolution estimation) | emd_47091_fsc.xml | 10.3 KB | Display | FSC data file |
| Images | emd_47091.png | 134 KB | ||
| Masks | emd_47091_msk_1.map | 91.1 MB | Mask map | |
| Filedesc metadata | emd-47091.cif.gz | 8.4 KB | ||
| Others | emd_47091_half_map_1.map.gz emd_47091_half_map_2.map.gz | 71.6 MB 71.6 MB | ||
| Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-47091 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-47091 | HTTPS FTP |
-Validation report
| Summary document | emd_47091_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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| Full document | emd_47091_full_validation.pdf.gz | 1.1 MB | Display | |
| Data in XML | emd_47091_validation.xml.gz | 17.4 KB | Display | |
| Data in CIF | emd_47091_validation.cif.gz | 23 KB | Display | |
| Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-47091 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-47091 | HTTPS FTP |
-Related structure data
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Links
| EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Map
| File | Download / File: emd_47091.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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| Annotation | Sharpened map of Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription | ||||||||||||||||||||||||||||||||||||
| Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
| Voxel size | X=Y=Z: 0.9208 Å | ||||||||||||||||||||||||||||||||||||
| Density |
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| Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
| Details | EMDB XML:
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-Supplemental data
-Mask #1
| File | emd_47091_msk_1.map | ||||||||||||
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| Projections & Slices |
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| Density Histograms |
-Half map: Half-map 2 of Taeniopygia guttata R2 retrotransposon initiating...
| File | emd_47091_half_map_1.map | ||||||||||||
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| Annotation | Half-map 2 of Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription | ||||||||||||
| Projections & Slices |
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| Density Histograms |
-Half map: Half-map 1 of Taeniopygia guttata R2 retrotransposon initiating...
| File | emd_47091_half_map_2.map | ||||||||||||
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| Annotation | Half-map 1 of Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription | ||||||||||||
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| Density Histograms |
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Sample components
-Entire : Taeniopygia guttata R2 retrotransposon initiating target-primed r...
| Entire | Name: Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription |
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| Components |
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-Supramolecule #1: Taeniopygia guttata R2 retrotransposon initiating target-primed r...
| Supramolecule | Name: Taeniopygia guttata R2 retrotransposon initiating target-primed reverse transcription type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 380 KDa |
-Macromolecule #1: R2Tg retrotransposon ORF
| Macromolecule | Name: R2Tg retrotransposon ORF / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 157.228484 KDa |
| Recombinant expression | Organism: ![]() |
| Sequence | String: MASCPKPGPP VSAGAMSLES GLTTHSVLAI ERGPNSLANS GSDFGGGGLG LPLRLLRVSV GTQTSRSDWV DLVSWSHPGP TSKSQQVDL VSLFPKHRVD LLSKNDQVDL VAQFLPSKFP PNLAENDLAL LVNLEFYRSD LHVYECVHFA AHWEGLSGLP E VYEQLAPQ ...String: MASCPKPGPP VSAGAMSLES GLTTHSVLAI ERGPNSLANS GSDFGGGGLG LPLRLLRVSV GTQTSRSDWV DLVSWSHPGP TSKSQQVDL VSLFPKHRVD LLSKNDQVDL VAQFLPSKFP PNLAENDLAL LVNLEFYRSD LHVYECVHFA AHWEGLSGLP E VYEQLAPQ PCVGETLHSS LPRDSELFVP EEGSSEKESE DAPKTSPPTP GKHGLEQTGE EKVMVTVPDK NPPCPCCGTR VN SVLNLIE HLKVSHGKRG VCFRCAKCGK ENSNYHSVVC HFPKCRGPET EKAPAGEWIC EVCNRDFTTK IGLGQHKRLA HPA VRNQER IVASQPKETS NRGAHKRCWT KEEEELLIRL EAQFEGNKNI NKLIAEHITT KTAKQISDKR RLLSRKPAEE PREE PGTCH HTRRAAASLR TEPEMSHHAQ AEDRDNGPGR RPLPGRAAAG GRTMDEIRRH PDKGNGQQRP TKQKSEEQLQ AYYKK TLEE RLSAGALNTF PRAFKQVMEG RDIKLVINQT AQDCFGCLES ISQIRTATRD KKDTVTREKH PKKPFQKWMK DRAIKK GNY LRFQRLFYLD RGKLAKIILD DIECLSCDIP LSEIYSVFKT RWETTGSFKS LGDFKTYGKA DNTAFRELIT AKEIEKN VQ EMSKGSAPGP DGITLGDVVK MDPEFSRTME IFNLWLTTGK IPDMVRGCRT VLIPKSSKPD RLKDINNWRP ITIGSILL R LFSRIVTARL SKACPLNPRQ RGFIRAAGCS ENLKLLQTII WSAKREHRPL GVVFVDIAKA FDTVSHQHII HALQQREVD PHIVGLVSNM YENISTYITT KRNTHTDKIQ IRVGVKQGDP MSPLLFNLAM DPLLCKLEES GKGYHRGQSS ITAMAFADDL VLLSDSWEN MNTNISILET FCNLTGLKTQ GQKCHGFYIK PTKDSYTIND CAAWTINGTP LNMIDPGESE KYLGLQFDPW I GIARSGLS TKLDFWLQRI DQAPLKPLQK TDILKTYTIP RLIYIADHSE VKTALLETLD QKIRTAVKEW LHLPPCTCDA IL YSSTRDG GLGITKLAGL IPSVQARRLH RIAQSSDDTM KCFMEKEKME QLHKKLWIQA GGDRENIPSI WEAPPSSEPP NNV STNSEW EAPTQKDKFP KPCNWRKNEF KKWTKLASQG RGIVNFERDK ISNHWIQYYR RIPHRKLLTA LQLRANVYPT REFL ARGRQ DQYIKACRHC DADIESCAHI IGNCPVTQDA RIKRHNYICE LLLEEAKKKD WVVFKEPHIR DSNKELYKPD LIFVK DARA LVVDVTVRYE AAKSSLEEAA AEKVRKYKHL ETEVRHLTNA KDVTFVGFPL GARGKWHQDN FKLLTELGLS KSRQVK MAE TFSTVALFSS VDIVHMFASR ARKSMVM |
-Macromolecule #2: 28S DNA bottom strand, 3' side
| Macromolecule | Name: 28S DNA bottom strand, 3' side / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 65.051477 KDa |
| Sequence | String: (DT)(DT)(DC)(DC)(DC)(DT)(DT)(DG)(DG)(DC) (DT)(DG)(DT)(DG)(DG)(DT)(DT)(DT)(DC)(DG) (DC)(DT)(DA)(DG)(DA)(DT)(DA)(DG)(DT) (DA)(DG)(DA)(DT)(DA)(DG)(DG)(DG)(DA)(DC) (DA) (DG)(DT)(DG)(DG)(DG)(DA) ...String: (DT)(DT)(DC)(DC)(DC)(DT)(DT)(DG)(DG)(DC) (DT)(DG)(DT)(DG)(DG)(DT)(DT)(DT)(DC)(DG) (DC)(DT)(DA)(DG)(DA)(DT)(DA)(DG)(DT) (DA)(DG)(DA)(DT)(DA)(DG)(DG)(DG)(DA)(DC) (DA) (DG)(DT)(DG)(DG)(DG)(DA)(DA)(DT) (DC)(DT)(DC)(DG)(DT)(DT)(DA)(DA)(DT)(DC) (DC)(DA) (DT)(DT)(DC)(DA)(DT)(DG)(DC) (DG)(DC)(DG)(DT)(DC)(DA)(DC)(DT)(DA)(DA) (DT)(DT)(DA) (DG)(DA)(DT)(DG)(DA)(DC) (DG)(DA)(DG)(DG)(DC)(DA)(DT)(DT)(DT)(DG) (DG)(DC)(DT)(DA) (DC)(DC)(DT)(DT)(DA) (DA)(DG)(DA)(DG)(DA)(DG)(DT)(DC)(DA)(DT) (DA)(DG)(DT)(DT)(DA) (DC)(DT)(DC)(DC) (DC)(DG)(DC)(DC)(DG)(DT)(DT)(DT)(DA)(DC) (DC)(DC)(DG)(DC)(DG)(DC) (DT)(DT)(DC) (DA)(DT)(DT)(DG)(DA)(DA)(DT)(DT)(DT)(DC) (DT)(DT)(DC)(DA)(DC)(DT)(DT) (DT)(DG) (DA)(DC)(DA)(DT)(DT)(DC)(DA)(DG)(DA)(DG) (DC)(DA)(DC)(DT)(DG)(DG)(DG)(DC) (DA) (DG)(DA)(DA)(DA)(DT)(DC)(DA)(DC)(DA)(DT) (DC)(DG)(DC)(DG)(DT)(DC)(DA)(DA)(DC) (DC)(DA)(DG)(DC)(DT)(DG)(DA)(DA)(DA)(DA) (DA) |
-Macromolecule #3: 28S DNA bottom strand, 5' side (priming strand)
| Macromolecule | Name: 28S DNA bottom strand, 5' side (priming strand) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 3.997607 KDa |
| Sequence | String: (DG)(DG)(DC)(DA)(DT)(DT)(DT)(DG)(DG)(DC) (DT)(DA)(DT) |
-Macromolecule #5: 28S DNA top strand
| Macromolecule | Name: 28S DNA top strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 65.225641 KDa |
| Sequence | String: (DT)(DT)(DT)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DG)(DT)(DT)(DG)(DA)(DC)(DG)(DC)(DG) (DA)(DT)(DG)(DT)(DG)(DA)(DT)(DT)(DT) (DC)(DT)(DG)(DC)(DC)(DC)(DA)(DG)(DT)(DG) (DC) (DT)(DC)(DT)(DG)(DA)(DA) ...String: (DT)(DT)(DT)(DT)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DG)(DT)(DT)(DG)(DA)(DC)(DG)(DC)(DG) (DA)(DT)(DG)(DT)(DG)(DA)(DT)(DT)(DT) (DC)(DT)(DG)(DC)(DC)(DC)(DA)(DG)(DT)(DG) (DC) (DT)(DC)(DT)(DG)(DA)(DA)(DT)(DG) (DT)(DC)(DA)(DA)(DA)(DG)(DT)(DG)(DA)(DA) (DG)(DA) (DA)(DA)(DT)(DT)(DC)(DA)(DA) (DT)(DG)(DA)(DA)(DG)(DC)(DG)(DC)(DG)(DG) (DG)(DT)(DA) (DA)(DA)(DC)(DG)(DG)(DC) (DG)(DG)(DG)(DA)(DG)(DT)(DA)(DA)(DC)(DT) (DA)(DT)(DG)(DA) (DC)(DT)(DC)(DT)(DC) (DT)(DT)(DA)(DA)(DG)(DG)(DT)(DA)(DG)(DC) (DC)(DA)(DA)(DA)(DT) (DG)(DC)(DC)(DT) (DC)(DG)(DT)(DC)(DA)(DT)(DC)(DT)(DA)(DA) (DT)(DT)(DA)(DG)(DT)(DG) (DA)(DC)(DG) (DC)(DG)(DC)(DA)(DT)(DG)(DA)(DA)(DT)(DG) (DG)(DA)(DT)(DT)(DA)(DA)(DC) (DG)(DA) (DG)(DA)(DT)(DT)(DC)(DC)(DC)(DA)(DC)(DT) (DG)(DT)(DC)(DC)(DC)(DT)(DA)(DT) (DC) (DT)(DA)(DC)(DT)(DA)(DT)(DC)(DT)(DA)(DG) (DC)(DG)(DA)(DA)(DA)(DC)(DC)(DA)(DC) (DA)(DG)(DC)(DC)(DA)(DA)(DG)(DG)(DG)(DA) (DA) |
-Macromolecule #4: R2Tg 3'UTR RNA
| Macromolecule | Name: R2Tg 3'UTR RNA / type: rna / ID: 4 / Number of copies: 1 |
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| Source (natural) | Organism: ![]() |
| Molecular weight | Theoretical: 106.656273 KDa |
| Sequence | String: UUCAGGUUAU UUAGAUGCUU AGUUUUUGUA CCUUUCUUGU UUUGUUUAGG AUUUUGAUAG UGUUAGUAUU UUUAUAUUUU UGUACGAUU GCAUAAUGUU CUUUUUUAUA CAGUUCUGUU UUAAUAAAAU AGACGAUAGC UAGAGACGUU AGGGCAGCCA C AAGCCAGU ...String: UUCAGGUUAU UUAGAUGCUU AGUUUUUGUA CCUUUCUUGU UUUGUUUAGG AUUUUGAUAG UGUUAGUAUU UUUAUAUUUU UGUACGAUU GCAUAAUGUU CUUUUUUAUA CAGUUCUGUU UUAAUAAAAU AGACGAUAGC UAGAGACGUU AGGGCAGCCA C AAGCCAGU UAGGUAGCGG AUAGUAGGUA GGAACAGACU UUUACUAUUU CAUAACGCGU CAAUUACCAC CUGAUUUGGA CC AAUUCAC GGGAUUUGUC CAAGGUGGAC GGGCCACCUU UACUUAACCC GGAAAAGGAA CAUAUAUAAU UUAUGUGUGU UCG AUAAAU AGCCAAA |
-Macromolecule #6: MAGNESIUM ION
| Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG |
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| Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: THYMIDINE-5'-TRIPHOSPHATE
| Macromolecule | Name: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: TTP |
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| Molecular weight | Theoretical: 482.168 Da |
| Chemical component information | ![]() ChemComp-TTP: |
-Macromolecule #8: ZINC ION
| Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: ZN |
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| Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
| Method | cryo EM |
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Processing | single particle reconstruction |
| Aggregation state | particle |
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Sample preparation
| Buffer | pH: 7.9 Details: 20 mM HEPES-KOH pH 7.9, 400 mM potassium acetate, 5 mM magnesium acetate, 0.1 mM dTTP |
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| Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. |
| Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285 K / Instrument: FEI VITROBOT MARK IV |
| Details | A260 = 17 |
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Electron microscopy
| Microscope | FEI TITAN KRIOS |
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| Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
| Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10627 / Average exposure time: 1.02 sec. / Average electron dose: 59.6 e/Å2 |
| Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
| Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000 |
| Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
| Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
| Refinement | Protocol: AB INITIO MODEL |
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| Output model | ![]() PDB-9dou: |
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Authors
United States, 1 items
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FIELD EMISSION GUN

