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- EMDB-49517: Structure of R2 retrotransposon protein from Platysternon megacep... -

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Basic information

Entry
Database: EMDB / ID: EMD-49517
TitleStructure of R2 retrotransposon protein from Platysternon megacephalum after second strand nicking
Map data
Sample
  • Complex: R2 retrotransposon protein after second strand nicking
    • Protein or peptide: R2 retrotransposon protein
    • DNA: Bottom strand of target rDNA
    • DNA: complementary DNA
    • RNA: 3'UTR RNA
    • DNA: Top strand for target rDNA
  • Ligand: ZINC ION
KeywordsRetrotransposon / Reverse transcriptase / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesPlatysternon megacephalum (big-headed turtle) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 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_49517.png

Downloads & links

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Map

FileDownload / File: emd_49517.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.)
1.14 Å/pix.
x 224 pix.
= 255.36 Å		1.14 Å/pix.
x 224 pix.
= 255.36 Å		1.14 Å/pix.
x 224 pix.
= 255.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0152
Minimum - Maximum-0.018005596 - 0.051070973
Average (Standard dev.)0.00023892455 (±0.002015374)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 255.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49517_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_49517_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 after second strand nicking

EntireName: R2 retrotransposon protein after second strand nicking
Components
  • Complex: R2 retrotransposon protein after second strand nicking
    • Protein or peptide: R2 retrotransposon protein
    • DNA: Bottom strand of target rDNA
    • DNA: complementary DNA
    • RNA: 3'UTR RNA
    • DNA: Top strand for target rDNA
  • Ligand: ZINC ION

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Supramolecule #1: R2 retrotransposon protein after second strand nicking

SupramoleculeName: R2 retrotransposon protein after second strand nicking
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Platysternon megacephalum (big-headed turtle)
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: Platysternon megacephalum (big-headed turtle)
Molecular weightTheoretical: 127.729367 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QKTIIQLPND NPACPFCGDH VGKPSALNVH LKRNHGGREV EFQCSMCNKA DPKAHSILCH IPKCKGKVTE EPTGDWACET CNKQFNTKS GLSQHKRIAH PAIRNQERIA ASQPKPNSQR GKHNSCWTVE EEQLLAAFNN MFWGKKNINI LISDHIHMKT A KQISEKRR ...String:
QKTIIQLPND NPACPFCGDH VGKPSALNVH LKRNHGGREV EFQCSMCNKA DPKAHSILCH IPKCKGKVTE EPTGDWACET CNKQFNTKS GLSQHKRIAH PAIRNQERIA ASQPKPNSQR GKHNSCWTVE EEQLLAAFNN MFWGKKNINI LISDHIHMKT A KQISEKRR LLGLNKNATV TTTNPLPVSS TCHLKIRTDS PNTTTGLKDT YMCKINENIV NQGQIKFDSE VISAWMAGDS NI RSLVEST SLDILSTFLM ETPKPRKKGN NKITNKKSGK KKKWMEKRAV KKGFYKRYQH LFETDRCKLA SIILDGTERL QCQ IPLTEI LETYKSKWET LTPFEGLGQF KSHAVADNTA FEILLSAKEI MKNIKEMNKN SAPGPDKVSL RDLLLADPEC NALE KLFNT WLITGIIPNS IKECRSLLIP KTADPEALKE LGNWRPLTIG SIVLRLFSRI ITNRLAKACP INARQRGFIA TPGCS ENLK ILHTIVKQAK TSKKSLGVVF VDIAKAFDSV SHDHIMWVLQ ERGLDQHIVN IIEDSYKKIH TRMEVGTERT PPIEIK VGV KQGDPMSPLL FNLAIDPLIT ALEKANTGFS YGKNKITSLA FADDLVMLSD TWEGMNKNIQ ILETFCNLSG LKVQAKK CY GFFLSPTHDS YTINKCDAWK IDKDSLNMIQ PGESEKYLGL KVDPWIGFSK PVLAEKLTIW LKRLTEAPLK PSQKLTML N IYTIPRIIYL ADHTDTKKTL LSSLDDNIRT VVKGWLHLPP DTCNGFIYTK TRDGGLGVTR LASLIPSIQA RRLHRIATS EDETIRNIAM ANNIEEEFQN LWVTAGGKKE EIPRITDPVS IDYRLPRRIL ELLNEWEKPA PKKMYPIPCN WREAEMAHWK NLPCQGSGI EHFDNDTISN DWLQFHRGFS ERQFLMGLKI RANVYPTREY QGRGRTNKNV NCRNCTASYE SLSHILGQCP A VQGARIRR HNKLCSMLKR EAKELKWVVY EEPHLHTTEK ELRKPDLIFV KEEMALVVDV TVRFEYKEKV FEDAAAEKVR HY KDLTSQI KELTGAKEIE YFGFPLGARG KWPEINEKVL TALGMPDYQQ KRTAKRFSKR TLLYSIDVIN TFENIGKNNK NNV P

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Macromolecule #2: Bottom strand of target rDNA

MacromoleculeName: Bottom strand of 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: complementary DNA

MacromoleculeName: complementary DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 22.722555 KDa
SequenceString: (DG)(DG)(DC)(DT)(DA)(DT)(DT)(DT)(DT)(DC) (DC)(DG)(DA)(DA)(DC)(DA)(DC)(DA)(DT)(DA) (DT)(DA)(DA)(DT)(DT)(DA)(DA)(DT)(DA) (DT)(DA)(DT)(DG)(DT)(DT)(DC)(DC)(DT)(DT) (DT) (DT)(DC)(DC)(DG)(DG)(DG) ...String:
(DG)(DG)(DC)(DT)(DA)(DT)(DT)(DT)(DT)(DC) (DC)(DG)(DA)(DA)(DC)(DA)(DC)(DA)(DT)(DA) (DT)(DA)(DA)(DT)(DT)(DA)(DA)(DT)(DA) (DT)(DA)(DT)(DG)(DT)(DT)(DC)(DC)(DT)(DT) (DT) (DT)(DC)(DC)(DG)(DG)(DG)(DT)(DT) (DA)(DA)(DG)(DT)(DA)(DA)(DA)(DG)(DG)(DT) (DG)(DG) (DC)(DC)(DC)(DG)(DT)(DC)(DC) (DA)(DC)(DC)(DT)(DT)(DG)(DC)

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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: 23.837215 KDa
SequenceString:
GCAAGGUGGA CGGGCCACCU UUACUUAACC CGGAAAAGGA ACAUAUAUUA AUUAUAUGUG UUCGGAAAAU AGCC

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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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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 TALOS
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: 9195 / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 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: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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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: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 32239
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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