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- EMDB-49516: Structure of R2 retrotransposon protein from Taeniopygia guttata ... -

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Basic information

Entry
Database: EMDB / ID: EMD-49516
TitleStructure of R2 retrotransposon protein from Taeniopygia guttata initiating target-primed reverse transcription
Map data
Sample
  • Complex: R2 retrotransposon protein in TPRT initiation stage
    • Protein or peptide: R2 retrotransposon protein
    • DNA: Bottom strand for target rDNA
    • DNA: Primer
    • RNA: 3'UTR RNA
    • DNA: Top strand for target rDNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
KeywordsRetrotransposon / Reverse transcriptase / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesTaeniopygia guttata (zebra finch) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsThawani A / Collins K / Nogales E
Funding support United States, 2 items
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, 2025-
Header (metadata) releaseJun 18, 2025-
Map releaseJun 18, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49516.png

Downloads & links

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Map

FileDownload / File: emd_49516.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 224 pix.
= 234.752 Å		1.05 Å/pix.
x 224 pix.
= 234.752 Å		1.05 Å/pix.
x 224 pix.
= 234.752 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0256
Minimum - Maximum-0.062972754 - 0.1316263
Average (Standard dev.)0.00029006152 (±0.0038644671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 234.752 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49516_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_49516_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : R2 retrotransposon protein in TPRT initiation stage

EntireName: R2 retrotransposon protein in TPRT initiation stage
Components
  • Complex: R2 retrotransposon protein in TPRT initiation stage
    • Protein or peptide: R2 retrotransposon protein
    • DNA: Bottom strand for target rDNA
    • DNA: Primer
    • RNA: 3'UTR RNA
    • DNA: Top strand for target rDNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: R2 retrotransposon protein in TPRT initiation stage

SupramoleculeName: R2 retrotransposon protein in TPRT initiation stage / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: R2 retrotransposon protein

MacromoleculeName: R2 retrotransposon protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Taeniopygia guttata (zebra finch)
Molecular weightTheoretical: 133.494391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVTVPDKNPP CPCCGTRVNS VLNLIEHLKV SHGKRGVCFR CAKCGKENSN YHSVVCHFPK CRGPETEKAP AGEWICEVCN RDFTTKIGL GQHKRLAHPA VRNQERIVAS QPKETSNRGA HKRCWTKEEE ELLIRLEAQF EGNKNINKLI AEHITTKTAK Q ISDKRRLL ...String:
MVTVPDKNPP CPCCGTRVNS VLNLIEHLKV SHGKRGVCFR CAKCGKENSN YHSVVCHFPK CRGPETEKAP AGEWICEVCN RDFTTKIGL GQHKRLAHPA VRNQERIVAS QPKETSNRGA HKRCWTKEEE ELLIRLEAQF EGNKNINKLI AEHITTKTAK Q ISDKRRLL SRKPAEEPRE EPGTCHHTRR AAASLRTEPE MSHHAQAEDR DNGPGRRPLP GRAAAGGRTM DEIRRHPDKG NG QQRPTKQ KSEEQLQAYY KKTLEERLSA GALNTFPRAF KQVMEGRDIK LVINQTAQDC FGCLESISQI RTATRDKKDT VTR EKHPKK PFQKWMKDRA IKKGNYLRFQ RLFYLDRGKL AKIILDDIEC LSCDIPLSEI YSVFKTRWET TGSFKSLGDF KTYG KADNT AFRELITAKE IEKNVQEMSK GSAPGPDGIT LGDVVKMDPE FSRTMEIFNL WLTTGKIPDM VRGCRTVLIP KSSKP DRLK DINNWRPITI GSILLRLFSR IVTARLSKAC PLNPRQRGFI RAAGCSENLK LLQTIIWSAK REHRPLGVVF VDIAKA FDT VSHQHIIHAL QQREVDPHIV GLVSNMYENI STYITTKRNT HTDKIQIRVG VKQGDPMSPL LFNLAMDPLL CKLEESG KG YHRGQSSITA MAFADDLVLL SDSWENMNTN ISILETFCNL TGLKTQGQKC HGFYIKPTKD SYTINDCAAW TINGTPLN M IDPGESEKYL GLQFDPWIGI ARSGLSTKLD FWLQRIDQAP LKPLQKTDIL KTYTIPRLIY IADHSEVKTA LLETLDQKI RTAVKEWLHL PPCTCDAILY SSTRDGGLGI TKLAGLIPSV QARRLHRIAQ SSDDTMKCFM EKEKMEQLHK KLWIQAGGDR ENIPSIWEA PPSSEPPNNV STNSEWEAPT QKDKFPKPCN WRKNEFKKWT KLASQGRGIV NFERDKISNH WIQYYRRIPH R KLLTALQL RANVYPTREF LARGRQDQYI KACRHCDADI ESCAHIIGNC PVTQDARIKR HNYICELLLE EAKKKDWVVF KE PHIRDSN KELYKPDLIF VKDARALVVD VTVRYEAAKS SLEEAAAEKV RKYKHLETEV RHLTNAKDVT FVGFPLGARG KWH QDNFKL LTELGLSKSR QVKMAETFST VALFSSVDIV HMFASRARKS MVM

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Macromolecule #2: Bottom strand for target rDNA

MacromoleculeName: Bottom strand for target rDNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.507758 KDa
SequenceString: (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) ...String:
(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) (DC)(DA)(DG)

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Macromolecule #3: Primer

MacromoleculeName: Primer / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.997607 KDa
SequenceString:
(DG)(DG)(DC)(DA)(DT)(DT)(DT)(DG)(DG)(DC) (DT)(DA)(DT)

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Macromolecule #5: Top strand for target rDNA

MacromoleculeName: Top strand for target rDNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.654875 KDa
SequenceString: (DC)(DT)(DG)(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) ...String:
(DC)(DT)(DG)(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)

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Macromolecule #4: 3'UTR RNA

MacromoleculeName: 3'UTR RNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 95.589312 KDa
SequenceString: UAGGGUAGAU AAUCUUUGUA UAGUGGGGGG GGAUCUCAUG UACCGGGUUU CUUUUAUUUG AUUUUCAAUA AAACAGACGG UAGCUAGGU UCGCAAGGCA GCCACAAGCC AAAGAUAGGU AGGGUGCUCA UAGUGAGUAG GGACAGUGCC UUUUGAUUCA C AACGCGUC ...String:
UAGGGUAGAU AAUCUUUGUA UAGUGGGGGG GGAUCUCAUG UACCGGGUUU CUUUUAUUUG AUUUUCAAUA AAACAGACGG UAGCUAGGU UCGCAAGGCA GCCACAAGCC AAAGAUAGGU AGGGUGCUCA UAGUGAGUAG GGACAGUGCC UUUUGAUUCA C AACGCGUC AAUACCAUCU GACACGGAUA CCCUUACCGG ACUUGUCAUG AUCUCCCAGA CUUGUCCAAG GUGGACGGGC CA CCUUUAC UUAACCCGGA AAAGGAACAU AUAUUAAUUA UAUGUGUUCG GAAAAUAGCC

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 5096 / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: RELION SGD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 18892
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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