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- EMDB-35348: Structure of R2 with 3'UTR and DNA in unwinding state -

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Basic information

Entry
Database: EMDB / ID: EMD-35348
TitleStructure of R2 with 3'UTR and DNA in unwinding state
Map data
Sample
  • Complex: R2 complex
    • Protein or peptide: Reverse transcriptase-like protein
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
    • RNA: 3'UTR
  • Ligand: ZINC ION
KeywordsR2 complex / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyZinc finger C2H2 type domain profile. / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / RNA-directed DNA polymerase activity / DNA/RNA polymerase superfamily / Reverse transcriptase-like protein
Function and homology information
Biological speciesBombyx mori (domestic silkworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsDeng P / Tan S / Wang J / Liu JJ
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Cell / Year: 2023
Title: Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon.
Authors: Pujuan Deng / Shun-Qing Tan / Qi-Yu Yang / Liangzheng Fu / Yachao Wu / Han-Zhou Zhu / Lei Sun / Zhangbin Bao / Yi Lin / Qiangfeng Cliff Zhang / Haoyi Wang / Jia Wang / Jun-Jie Gogo Liu /
Abstract: Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of ...Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.
History
DepositionFeb 10, 2023-
Header (metadata) releaseSep 20, 2023-
Map releaseSep 20, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_35348.png

Downloads & links

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Map

FileDownload / File: emd_35348.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0979 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.8316901 - 1.5718646
Average (Standard dev.)0.0018240119 (±0.057677764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 219.58 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35348_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_35348_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : R2 complex

EntireName: R2 complex
Components
  • Complex: R2 complex
    • Protein or peptide: Reverse transcriptase-like protein
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
    • RNA: 3'UTR
  • Ligand: ZINC ION

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Supramolecule #1: R2 complex

SupramoleculeName: R2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 177.28 KDa

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Macromolecule #1: Reverse transcriptase-like protein

MacromoleculeName: Reverse transcriptase-like protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 123.36243 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMASTALSLM GRCNPDGCTR GKHVTAAPMD GPRGPSSLAG TFGWGLAIPA GEPCGRVCSP ATVGFFPVAK KSNKENRPEA SGLPLESER TGDNPTVRGS AGADPVGQDA PGWTCQFCER TFSTNRGLGV HKRRAHPVET NTDAAPMMVK RRWHGEEIDL L ARTEARLL ...String:
MMASTALSLM GRCNPDGCTR GKHVTAAPMD GPRGPSSLAG TFGWGLAIPA GEPCGRVCSP ATVGFFPVAK KSNKENRPEA SGLPLESER TGDNPTVRGS AGADPVGQDA PGWTCQFCER TFSTNRGLGV HKRRAHPVET NTDAAPMMVK RRWHGEEIDL L ARTEARLL AERGQCSGGD LFGALPGFGR TLEAIKGQRR REPYRALVQA HLARFGSQPG PSSGGCSAEP DFRRASGAEE AG EERCAED AAAYDPSAVG QMSPDAARVL SELLEGAGRR RACRAMRPKT AGRRNDLHDD RTASAHKTSR QKRRAEYARV QEL YKKCRS RAAAEVIDGA CGGVGHSLEE METYWRPILE RVSDAPGPTP EALHALGRAE WHGGNRDYTQ LWKPISVEEI KASR FDWRT SPGPDGIRSG QWRAVPVHLK AEMFNAWMAR GEIPEILRQC RTVFVPKVER PGGPGEYRPI SIASIPLRHF HSILA RRLL ACCPPDARQR GFICADGTLE NSAVLDAVLG DSRKKLRECH VAVLDFAKAF DTVSHEALVE LLRLRGMPEQ FCGYIA HLY DTASTTLAVN NEMSSPVKVG RGVRQGDPLS PILFNVVMDL ILASLPERVG YRLEMELVSA LAYAYDLVLL AGSKVGM QE SISAVDCVGR QMGLRLNCRK SAVLSMIPDG HRKKHHYLTE RTFNIGGKPL RQVSCVERWR YLGVDFEASG CVTLEHSI S SALNNISRAP LKPQQRLEIL RAHLIPRFQH GFVLGNISDD RLRMLDVQIR KAVGQWLRLP ADVPKAYYHA AVQDGGLAI PSVRATIPDL IVRRFGGLDS SPWSVARAAA KSDKIRKKLR WAWKQLRRFS RVDSTTQRPS VRLFWREHLH ASVDGRELRE STRTPTSTK WIRERCAQIT GRDFVQFVHT HINALPSRIR GSRGRRGGGE SSLTCRAGCK VRETTAHILQ QCHRTHGGRI L RHNKIVSF VAKAMEENKW TVELEPRLRT SVGLRKPAII ASRDGVGVIV DVQVVSGQRS LDELHREKRN KYGNHGELVE LV AGRLGLP KAECVRATSC TISWRGVWSL TSYKELRSII GLREPTLQIV PILALRGSHM NWTRFNQMTS VMGGGVG

UniProtKB: Reverse transcriptase-like protein

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Macromolecule #2: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 18.434783 KDa
SequenceString: (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) ...String:
(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)(DG)

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Macromolecule #3: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 18.550889 KDa
SequenceString: (DC)(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) ...String:
(DC)(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)

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Macromolecule #4: 3'UTR

MacromoleculeName: 3'UTR / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 16.048569 KDa
SequenceString:
GUAGAUCAGG CCCGUCUGAU CCAAUUUCGC CGGCGUACCC GGCGAUGAAA

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / 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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: ab-initial in cryosparc
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3.31)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.31)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 106634

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-8ibx:
Structure of R2 with 3'UTR and DNA in unwinding state

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