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- EMDB-42637: Human LINE-1 retrotransposon ORF2 protein engaged with template R... -

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Basic information

Entry
Database: EMDB / ID: EMD-42637
TitleHuman LINE-1 retrotransposon ORF2 protein engaged with template RNA in elongation state
Map dataHuman LINE-1 retrotransposon ORF2 protein engaged with template RNA in elongation state
Sample
  • Complex: Human LINE-1 retrotransposon ORF2 protein in elongation stage
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
    • RNA: Template RNA
    • DNA: Complementary DNA
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
KeywordsLINE-1 / retrotransposon / reverse transcriptase / retroelement / RNA BINDING PROTEIN / RNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


retrotransposition / nucleic acid metabolic process / type II site-specific deoxyribonuclease activity / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / DNA recombination / RNA binding / metal ion binding
Similarity search - Function
Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
LINE-1 retrotransposable element ORF2 protein
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsThawani A / Florez Ariza AJ / 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)DP1HL156819 United States
CitationJournal: Nature / Year: 2024
Title: Template and target-site recognition by human LINE-1 in retrotransposition.
Authors: Akanksha Thawani / Alfredo Jose Florez Ariza / Eva Nogales / Kathleen Collins /
Abstract: The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease. L1 ...The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.
History
DepositionNov 6, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42637.png

Downloads & links

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Map

FileDownload / File: emd_42637.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman LINE-1 retrotransposon ORF2 protein engaged with template RNA in elongation state
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 0.0095
Minimum - Maximum-0.04018866 - 0.07824448
Average (Standard dev.)0.0003126624 (±0.0025608565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 194.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 2

Fileemd_42637_half_map_1.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_42637_half_map_2.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human LINE-1 retrotransposon ORF2 protein in elongation stage

EntireName: Human LINE-1 retrotransposon ORF2 protein in elongation stage
Components
  • Complex: Human LINE-1 retrotransposon ORF2 protein in elongation stage
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
    • RNA: Template RNA
    • DNA: Complementary DNA
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Human LINE-1 retrotransposon ORF2 protein in elongation stage

SupramoleculeName: Human LINE-1 retrotransposon ORF2 protein in elongation stage
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 175 KDa

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Macromolecule #1: LINE-1 retrotransposable element ORF2 protein

MacromoleculeName: LINE-1 retrotransposable element ORF2 protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 149.300391 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTGSNSHITI LTLNINGLNS AIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSTLDRSTRQ K VNKDTQEL ...String:
MTGSNSHITI LTLNINGLNS AIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSTLDRSTRQ K VNKDTQEL NSALHQADLI DIYRTLHPKS TEYTFFSAPH HTYSKIDHIV GSKALLSKCK RTEIITNYLS DHSAIKLELR IK NLTQSRS TTWKLNNLLL NDYWVHNEMK AEIKMFFETN ENKDTTYQNL WDAFKAVCRG KFIALNAHKR KQERSKIDTL TSQ LKELEK QEQTHSKASR RQEITKIRAE LKEIETQKTL QKINESRSWF FERINKIDRP LARLIKKKRE KNQIDTIKND KGDI TTDPT EIQTTIREYY KHLYANKLEN LEEMDKFLDT YTLPRLNQEE VESLNRPITG SEIVAIINSL PTKKSPGPDG FTAEF YQRY KEELVPFLLK LFQSIEKEGI LPNSFYEASI ILIPKPGRDT TKKENFRPIS LMNIDAKILN KILANRIQQH IKKLIH HDQ VGFIPGMQGW FNIRKSINVI QHINRAKDKN HMIISIDAEK AFDKIQQPFM LKTLNKLGID GTYFKIIRAI YDKPTAN II LNGQKLEAFP LKTGTRQGCP LSPLLFNIVL EVLARAIRQE KEIKGIQLGK EEVKLSLFAD DMIVYLENPI VSAQNLLK L ISNFSKVSGY KINVQKSQAF LYTNNRQTES QIMGELPFTI ASKRIKYLGI QLTRDVKDLF KENYKPLLKE IKEETNKWK NIPCSWVGRI NIVKMAILPK VIYRFNAIPI KLPMTFFTEL EKTTLKFIWN QKRARIAKSI LSQKNKAGGI TLPDFKLYYK ATVTKTAWY WYQNRDIDQW NRTEPSEIMP HIYNYLIFDK PEKNKQWGKD SLFNKWCWEN WLAICRKLKL DPFLTPYTKI N SRWIKDLN VKPKTIKTLE ENLGITIQDI GVGKDFMSKT PKAMATKDKI DKWDLIKLKS FCTAKETTIR VNRQPTTWEK IF ATYSSDK GLISRIYNEL KQIYKKKTNN PIKKWAKDMN RHFSKEDIYA AKKHMKKCSS SLAIREMQIK TTMRYHLTPV RMA IIKKSG NNRCWRGCGE IGTLLHCWWD CKLVQPLWKS VWRFLRDLEL EIPFDPAIPL LGIYPNEYKS CCYKDTCTRM FIAA LFTIA KTWNQPKCPT MIDWIKKMWH IYTMEYYAAI KNDEFISFVG TWMKLETIIL SKLSQEQKTK HRIFSLIGGN

UniProtKB: LINE-1 retrotransposable element ORF2 protein

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Macromolecule #2: Template RNA

MacromoleculeName: Template RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.956451 KDa
SequenceString:
GCAGACUGCG UAGGCGUGCA GGAACCUGCA CGCCUACGCA GUCUGCAAAA AAAAAAAAAA ACAAUAUCGG CGCG

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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: 3.951586 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DC)(DC)(DG)(DA)(DT)(DA) (DT)(DT)(DDG)

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Macromolecule #4: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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

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

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 2 / Number real images: 23878 / Average exposure time: 1.0 sec. / 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: INSILICO MODEL / In silico model: RELION SGD
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 111564
FSC plot (resolution estimation)

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