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- EMDB-40033: Bombyx mori R2 retrotransposon initiating target-primed reverse t... -

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Basic information

Entry
Database: EMDB / ID: EMD-40033
TitleBombyx mori R2 retrotransposon initiating target-primed reverse transcription
Map dataSharpened map of Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
Sample
  • Complex: Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
    • Protein or peptide: Reverse transcriptase-like protein
    • DNA: 28S DNA bottom strand, 3' side
    • DNA: 28S DNA bottom strand, 5' side (priming strand)
    • RNA: R2Bm 3'UTR RNA
    • DNA: 28S DNA top strand
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
Keywordsretrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN / 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: 3.08 Å
AuthorsWilkinson ME / Zhang F
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01HG009761 United States
CitationJournal: Science / Year: 2023
Title: Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription.
Authors: Max E Wilkinson / Chris J Frangieh / Rhiannon K Macrae / Feng Zhang /
Abstract: Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse ...Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is unwound at the insertion site and recognized by an upstream motif. An extension of the reverse transcriptase (RT) domain recognizes the retrotransposon RNA and guides the 3' end into the RT active site to template reverse transcription. We used Cas9 to retarget R2 in vitro to non-native sequences, suggesting future use as a reprogrammable RNA-based gene-insertion tool.
History
DepositionMar 9, 2023-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40033.png

Downloads & links

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Map

FileDownload / File: emd_40033.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
Voxel sizeX=Y=Z: 0.884 Å
Density
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.035769425 - 0.06533652
Average (Standard dev.)0.000040435985 (±0.0020603824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 265.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40033_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map one

Fileemd_40033_half_map_1.map
AnnotationHalf map one
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map two

Fileemd_40033_half_map_2.map
AnnotationHalf map two
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bombyx mori R2 retrotransposon initiating target-primed reverse t...

EntireName: Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
Components
  • Complex: Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
    • Protein or peptide: Reverse transcriptase-like protein
    • DNA: 28S DNA bottom strand, 3' side
    • DNA: 28S DNA bottom strand, 5' side (priming strand)
    • RNA: R2Bm 3'UTR RNA
    • DNA: 28S DNA top strand
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Bombyx mori R2 retrotransposon initiating target-primed reverse t...

SupramoleculeName: Bombyx mori R2 retrotransposon initiating target-primed reverse transcription
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 230 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: 123.358352 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 LAYADDLVLL 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 SVGLRKPDII ASRDGVGVIV DVQVVSGQRS LDELHREKRN KYGNHGELVE LV AGRLGLP KAECVRATSC TISWRGVWSL TSYKELRSII GLREPTLQIV PILALRGSHM NWTRFNQMTS VMGGGVG

UniProtKB: Reverse transcriptase-like protein

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Macromolecule #2: 28S DNA bottom strand, 3' side

MacromoleculeName: 28S DNA bottom strand, 3' side / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bombyx mori (domestic silkworm)
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: 28S DNA bottom strand, 5' side (priming strand)

MacromoleculeName: 28S DNA bottom strand, 5' side (priming strand) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 7.439812 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)(DT)

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Macromolecule #5: 28S DNA top strand

MacromoleculeName: 28S DNA top strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bombyx mori (domestic silkworm)
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: R2Bm 3'UTR RNA

MacromoleculeName: R2Bm 3'UTR RNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 81.21393 KDa
SequenceString: GCCUUGCACA GUAGUCCAGC GGUAAGGGUG UAGAUCAGGC CCGUCUGUUU CUCCCCCGGA GCUCGCUCCC UUGGCUUCCC UUAUAUAUU UUAACAUCAG AAACAGACAU UAAACAUCUA CUGAUCCAAU UUCGCCGGCG UACGGCCACG AUCGGGAGGG U GGGAAUCU ...String:
GCCUUGCACA GUAGUCCAGC GGUAAGGGUG UAGAUCAGGC CCGUCUGUUU CUCCCCCGGA GCUCGCUCCC UUGGCUUCCC UUAUAUAUU UUAACAUCAG AAACAGACAU UAAACAUCUA CUGAUCCAAU UUCGCCGGCG UACGGCCACG AUCGGGAGGG U GGGAAUCU CGGGGGUCUU CCGAUCCUAA UCCAUGAUGA UUACGACCUG AGUCACUAAA GACGAUGGCA UGAUGAUCCG GC GAUGAAA AUAGCC

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / 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 / Thymidine 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

BufferpH: 7.9
Details: 20 mM HEPES-KOH pH 7.9, 500 mM potassium acetate, 5 mM magnesium acetate, 1 mM TCEP
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285 K / Instrument: FEI VITROBOT MARK IV
DetailsA260 = 3

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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: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 16551 / Average exposure time: 0.69 sec. / Average electron dose: 42.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1085471
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Avg.num./class: 100000 / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 39616
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8gh6:
Bombyx mori R2 retrotransposon initiating target-primed reverse transcription

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