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Yorodumi- PDB-8uw3: Human LINE-1 retrotransposon ORF2 protein engaged with template R... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8uw3 | |||||||||
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Title | Human LINE-1 retrotransposon ORF2 protein engaged with template RNA in elongation state | |||||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA/DNA / LINE-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 | |||||||||
Biological species | Homo sapiens (human) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Thawani, A. / Florez Ariza, A.J. / Collins, K. / Nogales, E. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8uw3.cif.gz | 307.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8uw3.ent.gz | 238.2 KB | Display | PDB format |
PDBx/mmJSON format | 8uw3.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8uw3_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8uw3_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8uw3_validation.xml.gz | 48.2 KB | Display | |
Data in CIF | 8uw3_validation.cif.gz | 75.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uw/8uw3 ftp://data.pdbj.org/pub/pdb/validation_reports/uw/8uw3 | HTTPS FTP |
-Related structure data
Related structure data | 42637MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 149300.391 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O00370 |
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#2: RNA chain | Mass: 23956.451 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: DNA chain | Mass: 3951.586 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: Chemical | ChemComp-TTP / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Human LINE-1 retrotransposon ORF2 protein in elongation stage Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Value: 0.175 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 23878 |
Image scans | Width: 11520 / Height: 8184 |
-Processing
EM software | Name: RELION / Category: 3D reconstruction |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
Symmetry | Point symmetry: C1 (asymmetric) |
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111564 / Symmetry type: POINT |