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- PDB-9k6h: Structure of human LINE-1 ORF2p with endogenous DNA and RNA/cDNA ... -

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Basic information

Entry
Database: PDB / ID: 9k6h
TitleStructure of human LINE-1 ORF2p with endogenous DNA and RNA/cDNA hybrid bound to dNTP and Mn2+
Components
  • Complementary DNA
  • Genomic DNA (bottom strand)
  • Genomic DNA (top strand)
  • LINE-1 retrotransposable element ORF2 protein
  • Template RNA
KeywordsGENE REGULATION / Endonuclease / Reverse transcriptase
Function / homology
Function and homology information


nucleic acid metabolic process / retrotransposition / 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 domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / RNA / RNA (> 10) / LINE-1 retrotransposable element ORF2 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Trichoplusia ni (cabbage looper)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsJin, W. / Yu, C. / Xu, R.M.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32330021 China
National Natural Science Foundation of China (NSFC)92153302 China
Chinese Academy of SciencesXDB3701010 China
CitationJournal: Science / Year: 2025
Title: Mechanism of DNA targeting by human LINE-1.
Authors: Wenxing Jin / Cong Yu / Yan Zhang / Changchang Cao / Tianfan Xia / Ge Song / Zhaokui Cai / Yuanchao Xue / Bing Zhu / Rui-Ming Xu /
Abstract: Long interspersed nuclear element-1 (LINE-1 or L1), the only autonomously active retrotransposon in humans today, constitutes a large proportion of the genome and continues to evolve the genome and ...Long interspersed nuclear element-1 (LINE-1 or L1), the only autonomously active retrotransposon in humans today, constitutes a large proportion of the genome and continues to evolve the genome and impact fundamental biological processes. L1 retrotransposition critically depends on its endonuclease and reverse transcriptase subunit open reading frame 2 protein (ORF2p), which targets genomic loci and nicks DNA using an evolutionarily distinct yet not fully understood mechanism. Our structural and biochemical analyses revealed that ORF2p is a structure-dependent endonuclease. It binds a double-stranded DNA region upstream of the nicking site and recognizes a downstream forked or flap structure for efficient DNA nicking. This discovery suggests that L1 mobilization piggybacks on chromosomal processes with noncanonical DNA structure intermediates.
History
DepositionOct 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: LINE-1 retrotransposable element ORF2 protein
B: Genomic DNA (top strand)
C: Genomic DNA (bottom strand)
D: Complementary DNA
E: Template RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,1138
Polymers165,5025
Non-polymers6123
Water00
1


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

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Components

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DNA chain , 3 types, 3 molecules BCD

#2: DNA chain Genomic DNA (top strand)


Mass: 6026.977 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper)
#3: DNA chain Genomic DNA (bottom strand)


Mass: 3605.356 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper)
#4: DNA chain Complementary DNA


Mass: 3087.110 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper)

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Protein / RNA chain , 2 types, 2 molecules AE

#1: Protein LINE-1 retrotransposable element ORF2 protein / ORF2p


Mass: 149153.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: O00370, RNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#5: RNA chain Template RNA


Mass: 3629.032 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper)

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Non-polymers , 3 types, 3 molecules

#6: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human LINE-1 ORF2p with endogenous DNA and RNA/cDNA hybrid bound to dNTP and Mn2+COMPLEX#1-#2, #4, #3, #50RECOMBINANT
2Human LINE-1 ORF2pCOMPLEX#11RECOMBINANT
3endogenous DNA and RNA/cDNA hybridCOMPLEX#2-#51NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Trichoplusia ni (cabbage looper)7111
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7 / Details: 20 mM HEPES pH 7.0, 500 mM NaCl, 1 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 268644 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039970
ELECTRON MICROSCOPYf_angle_d0.55513672
ELECTRON MICROSCOPYf_dihedral_angle_d16.8011782
ELECTRON MICROSCOPYf_chiral_restr0.0391521
ELECTRON MICROSCOPYf_plane_restr0.0031514

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