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- EMDB-40859: Structure of LINE-1 ORF2p with an oligo(A) template -

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Basic information

Entry
Database: EMDB / ID: EMD-40859
TitleStructure of LINE-1 ORF2p with an oligo(A) template
Map data
Sample
  • Complex: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
    • RNA: RNA (25-MER)
Keywordsreverse transcriptase / LINE-1 / RNA BINDING PROTEIN-RNA 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: 3.66 Å
Authorsvan Eeuwen T / Taylor MS / Rout MP
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 United States
CitationJournal: Nature / Year: 2024
Title: Structures, functions and adaptations of the human LINE-1 ORF2 protein.
Authors: Eric T Baldwin / Trevor van Eeuwen / David Hoyos / Arthur Zalevsky / Egor P Tchesnokov / Roberto Sánchez / Bryant D Miller / Luciano H Di Stefano / Francesc Xavier Ruiz / Matthew Hancock / ...Authors: Eric T Baldwin / Trevor van Eeuwen / David Hoyos / Arthur Zalevsky / Egor P Tchesnokov / Roberto Sánchez / Bryant D Miller / Luciano H Di Stefano / Francesc Xavier Ruiz / Matthew Hancock / Esin Işik / Carlos Mendez-Dorantes / Thomas Walpole / Charles Nichols / Paul Wan / Kirsi Riento / Rowan Halls-Kass / Martin Augustin / Alfred Lammens / Anja Jestel / Paula Upla / Kera Xibinaku / Samantha Congreve / Maximiliaan Hennink / Kacper B Rogala / Anna M Schneider / Jennifer E Fairman / Shawn M Christensen / Brian Desrosiers / Gregory S Bisacchi / Oliver L Saunders / Nafeeza Hafeez / Wenyan Miao / Rosana Kapeller / Dennis M Zaller / Andrej Sali / Oliver Weichenrieder / Kathleen H Burns / Matthias Götte / Michael P Rout / Eddy Arnold / Benjamin D Greenbaum / Donna L Romero / John LaCava / Martin S Taylor /
Abstract: The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open ...The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p). ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer, autoimmunity and ageing, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.
History
DepositionMay 24, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40859.png

Downloads & links

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Map

FileDownload / File: emd_40859.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.179
Minimum - Maximum-0.0017836808 - 2.0167415
Average (Standard dev.)0.0015672902 (±0.02946434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40859_msk_1.map
Projections & Slices
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Additional map: #1

Fileemd_40859_additional_1.map
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Additional map: #2

Fileemd_40859_additional_2.map
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Half map: #1

Fileemd_40859_half_map_1.map
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Half map: #2

Fileemd_40859_half_map_2.map
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Sample components

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Entire : Long Interspersed Nuclear Element (LINE)-1 ORF2 protein

EntireName: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
Components
  • Complex: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
    • RNA: RNA (25-MER)

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Supramolecule #1: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein

SupramoleculeName: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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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 / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 149.252344 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSILDRSTRQ K VNKDTQEL ...String:
MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSILDRSTRQ K VNKDTQEL NSALHQTDLI DIYRTLHPKS TEYTFFSAPH HTYSKIDHIV GSKALLSKCK RTEIITNYLS DHSAIKLELR IK NLTQSRS TTWKLNNLLL NDYWVHNEMK AEIKMFFETN ENKDTTYQNL WDAFKAVCRG KFIALNAYKR KQERSKIDTL TSQ LKELEK QEQTHSKASR RQEITKIRAE LKEIETQKTL QKINESRSWF FERINKIDRP LARLIKKKRE KNQIDTIKND KGDI TTDPT EIQTTIREYY KHLYANKLEN LEEMDTFLDT YTLPRLNQEE VESLNRPITG SEIVAIINSL PTKKSPGPDG FTAEF YQRY KEELVPFLLK LFQSIEKEGI LPNSFYEASI ILIPKPGRDT TKKENFRPIS LMNIDAKILN KILANRIQQH IKKLIH HDQ VGFIPGMQGW FNIRKSINVI QHINRAKDKN HVIISIDAEK AFDKIQQPFM LKTLNKLGID GMYLKIIRAI YDKPTAN II LNGQKLEAFP LKTGTRQGCP LSPLLFNIVL EVLARAIRQE KEIKGIQLGK EEVKLSLFAD DMIVYLENPI VSAQNLLK L ISNFSKVSGY KINVQKSQAF LYNNNRQTES QIMGELPFTI ASKRIKYLGI QLTRDVKDLF KENYKPLLKE IKEDTNKWK NIPCSWVGRI NIVKMAILPK VIYRFNAIPI KLPMTFFTEL EKTTLKFIWN QKRARIAKSI LSQKNKAGGI TLPDFKLYYK ATVTKTAWY WYQNRDIDQW NRTEPSEIMP HIYNYLIFDK PEKNKQWGKD SLLNKWCWEN WLAICRKLKL DPFLTPYTKI N SRWIKDLN VKPKTIKTLE ENLGITIQDI GVGKDFMSKT PKAMATKDKI DKWDLIKLKS FCTAKETTIR VNRQPTTWEK IF ATYSSDK GLISRIYNEL KQIYKKKTNN PIKKWAKDMN RHFSKEDIYA AKKHMKKCSS SLAIREMQIK TTMRYHLTPV RMA IIKKSG NNRCWRGCGE IGTLVHCWWD CKLVQPLWKS VWRFLRDLEL EIPFDPAIPL LGIYPKDYKS CCYKDTCTRM FIAA LFTIA KTWNQPNCPT MIDWIKKMWH IYTMEYYAAI KNDEFISFVG TWMKLETIIL SKLSQEQKTK HRIFSLIGGN

UniProtKB: LINE-1 retrotransposable element ORF2 protein

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Macromolecule #2: RNA (25-MER)

MacromoleculeName: RNA (25-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.185188 KDa
SequenceString:
AAAAAAAAAA AAAAAAAAAA AAAAA

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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

Concentration0.15 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
2.0 mMMg(CH3COO)2magnesium acetate
2.0 mMC4H10O2S2DTT

Details: 20mM HEPES pH 7.6, 150mM NaCl, 2mM MgOAc, 2mM DTT, 2mM dTTP
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC / Details: Manually blotted from the back.
DetailsSample was monodisperse though unstable

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
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: 2 / Number real images: 11270 / Average electron dose: 54.0 e/Å2
Details: Counter mode images collected in dose-fractionation mode over 48 frames with a dose per frame of 1.16 e/A2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1536512
Details: Particles were template picked using 2D class average references
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model
Details: Ab initio model was generated in cryoSPARC from the data
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: cryoSPARC (ver. 3.3.1), RELION (ver. 3.1))
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 203314
DetailsMovies motion and gain corrected with MotionCorr2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8c8j


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 203
Output model

PDB-8sxu:
Structure of LINE-1 ORF2p with an oligo(A) template

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