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Open data
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Basic information
Entry | Database: PDB / ID: 8sxt | ||||||
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Title | Structure of LINE-1 ORF2p with template:primer hybrid | ||||||
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![]() | RNA BINDING PROTEIN/RNA/DNA / ![]() ![]() ![]() | ||||||
Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | van Eeuwen, T. / Taylor, M.S. / Rout, M.P. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 188.8 KB | Display | ![]() |
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PDB format | ![]() | 134.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 40858MC ![]() 8c8jC ![]() 8sxuC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: DNA chain | ![]() Mass: 3975.611 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#2: RNA chain | Mass: 5444.269 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: Protein | Mass: 149252.344 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() References: UniProt: O00370, ![]() ![]() |
#4: Chemical | ChemComp-TTP / ![]() |
#5: Chemical | ChemComp-MG / |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein Type: COMPLEX / Entity ID: #3 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Source (natural) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.6 Details: 20mM HEPES pH 7.6, 150mM NaCl, 2mM MgOAc, 2mM DTT, 2mM dTTP | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() | |||||||||||||||||||||||||
Specimen support | Details: Pelco easy glow | |||||||||||||||||||||||||
Vitrification![]() | Instrument: LEICA EM CPC / Cryogen name: ETHANE / Details: Manually blotted from the back |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11270 Details: Super-resolution images collected in dose-fractionation mode over 54 frames with a dose per frame of 1.08 e/A2 |
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Processing
EM software |
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Image processing | Details: Movies were binned during motion and gain correction | ||||||||||||||||||||||||||||||||||||||||
CTF correction![]() | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1074466 Details: Particles were template picked using 2D class average references | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry![]() | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 430198 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 109 / Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 8C8J Pdb chain-ID: A / Accession code: 8C8J / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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