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- PDB-8sxt: Structure of LINE-1 ORF2p with template:primer hybrid -

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Basic information

Entry
Database: PDB / ID: 8sxt
TitleStructure of LINE-1 ORF2p with template:primer hybrid
Components
  • DNA primerPrimer (molecular biology)
  • LINE-1 retrotransposable element ORF2 protein
  • RNA template
KeywordsRNA BINDING PROTEIN/RNA/DNA / reverse transcriptase / LINE-1 / RNA BINDING PROTEIN / 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
THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / LINE-1 retrotransposable element ORF2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
Authorsvan Eeuwen, T. / Taylor, M.S. / Rout, M.P.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: DNA primer
C: RNA template
A: LINE-1 retrotransposable element ORF2 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,1795
Polymers158,6723
Non-polymers5062
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain DNA primer / Primer (molecular biology)


Mass: 3975.611 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: RNA chain RNA template


Mass: 5444.269 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein LINE-1 retrotransposable element ORF2 protein / ORF2p


Mass: 149252.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
References: UniProt: O00370, RNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#4: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
Type: COMPLEX / Entity ID: #3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Details: 20mM HEPES pH 7.6, 150mM NaCl, 2mM MgOAc, 2mM DTT, 2mM dTTP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
32 mMmagnesium acetateMg(CH3COO)21
42 mMDTTC4H10O2S21
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse though unstable
Specimen supportDetails: Pelco easy glow
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 0 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron 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
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.1CTF correction
5CTFFIND4.1.13CTF correction
10cryoSPARC3.3.1initial Euler assignment
11RELION3.1initial Euler assignment
12cryoSPARC3.3.1final Euler assignment
14cryoSPARC3.3.13D reconstruction
15PHENIXmodel refinement
Image processingDetails: Movies were binned during motion and gain correction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1074466
Details: Particles were template picked using 2D class average references
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 430198 / Symmetry type: POINT
Atomic model buildingB value: 109 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 8C8J

8c8j


Pdb chain-ID: A / Accession code: 8C8J / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036555
ELECTRON MICROSCOPYf_angle_d0.5438932
ELECTRON MICROSCOPYf_dihedral_angle_d7.956986
ELECTRON MICROSCOPYf_chiral_restr0.038999
ELECTRON MICROSCOPYf_plane_restr0.0031050

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