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- PDB-9hdr: Human LINE-1 ORF2p target-primed reverse transcription complex wi... -

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Basic information

Entry
Database: PDB / ID: 9hdr
TitleHuman LINE-1 ORF2p target-primed reverse transcription complex with EN domain resolved
Components
  • (Target DNA strand ...) x 4
  • LINE-1 retrotransposable element ORF2 protein
  • Template P(A)30 RNA
  • Unassigned Nucleic Acid
KeywordsRNA BINDING PROTEIN / LINE-1 / L1 / ORF2p / Reverse transcriptase / endonuclease / DNA / RNA
Function / homology
Function and homology information


nucleic acid metabolic process / retrotransposition / SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / SUMOylation of transcription cofactors / Postmitotic nuclear pore complex (NPC) reformation / septin ring ...nucleic acid metabolic process / retrotransposition / SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / SUMOylation of transcription cofactors / Postmitotic nuclear pore complex (NPC) reformation / septin ring / SUMOylation of DNA damage response and repair proteins / type II site-specific deoxyribonuclease activity / Transcriptional and post-translational regulation of MITF-M expression and activity / SUMOylation of DNA replication proteins / SUMOylation of SUMOylation proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / SUMOylation of RNA binding proteins / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / SUMOylation of chromatin organization proteins / detection of maltose stimulus / maltose transport complex / carbohydrate transport / ubiquitin-like protein ligase binding / protein sumoylation / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / RNA-directed DNA polymerase activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / condensed nuclear chromosome / cell chemotaxis / RNA-directed DNA polymerase / protein tag activity / telomerase activity / outer membrane-bounded periplasmic space / DNA recombination / periplasmic space / DNA damage response / RNA binding / metal ion binding / identical protein binding / nucleus / membrane
Similarity search - Function
Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein ...Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / LINE-1 retrotransposable element ORF2 protein / Maltose/maltodextrin-binding periplasmic protein / Ubiquitin-like protein SMT3
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Saccharomyces cerevisiae (brewer's yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGhanim, G.E. / Hu, H. / Nguyen, T.H.D.
Funding support United Kingdom, United States, European Union, 3items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/19 United Kingdom
Jane Coffin Childs (JCC) Fund United States
European Molecular Biology Organization (EMBO)European Union
CitationJournal: Science / Year: 2025
Title: Structural mechanism of LINE-1 target-primed reverse transcription.
Authors: George E Ghanim / Hongmiao Hu / Jerome Boulanger / Thi Hoang Duong Nguyen /
Abstract: Long interspersed element-1 (LINE-1) retrotransposons are the only active autonomous transposable elements in humans. They propagate by reverse transcribing their messenger RNA into new genomic ...Long interspersed element-1 (LINE-1) retrotransposons are the only active autonomous transposable elements in humans. They propagate by reverse transcribing their messenger RNA into new genomic locations by a process called target-primed reverse transcription (TPRT). In this work, we present four cryo-electron microscopy structures of the human LINE-1 TPRT complex, revealing the conformational dynamics of open reading frame 2 protein (ORF2p) and its extensive remodeling of the target DNA for TPRT initiation. We observe nicking of the DNA second strand during reverse transcription of the first strand. Structure prediction identifies high-confidence binding sites for LINE-1-associated factors-namely proliferating cell nuclear antigen (PCNA) and cytoplasmic poly(A)-binding protein 1 (PABPC1)-on ORF2p. Together with our structural data, this suggests a mechanism by which these factors regulate retrotransposition and supports a model for TPRT that accounts for retrotransposition outcomes observed in cells.
History
DepositionNov 12, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2May 7, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: LINE-1 retrotransposable element ORF2 protein
B: Target DNA strand 1
C: Target DNA strand 2
D: Template P(A)30 RNA
E: Target DNA strand 3
F: Target DNA strand 4
G: Unassigned Nucleic Acid
hetero molecules


Theoretical massNumber of molelcules
Total (without water)252,68810
Polymers252,1327
Non-polymers5563
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Target DNA strand ... , 4 types, 4 molecules BCEF

#2: DNA chain Target DNA strand 1


Mass: 11424.341 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Modified from de novo LINE-1 insertion event into the FVIII gene
Source: (synth.) Homo sapiens (human)
#3: DNA chain Target DNA strand 2


Mass: 9144.987 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Modified from de novo LINE-1 insertion event into the FVIII gene
Source: (synth.) Homo sapiens (human)
#5: DNA chain Target DNA strand 3


Mass: 7900.112 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Modified from de novo LINE-1 insertion event into the FVIII gene
Source: (synth.) Homo sapiens (human)
#6: DNA chain Target DNA strand 4


Mass: 5905.973 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Modified from de novo LINE-1 insertion event into the FVIII gene
Source: (synth.) Homo sapiens (human)

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Protein / RNA chain / DNA chain , 3 types, 3 molecules ADG

#1: Protein LINE-1 retrotransposable element ORF2 protein / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / ORF2p


Mass: 206717.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast), (gene. exp.) Homo sapiens (human)
Cell line: High 5 / Gene: malE, b4034, JW3994, SMT3, YDR510W, D9719.15 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P0AEX9, UniProt: Q12306, UniProt: O00370, RNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#4: RNA chain Template P(A)30 RNA


Mass: 9831.217 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Chemically synthesized 30 nucleotide Poly(A) sequence
Source: (synth.) Homo sapiens (human)
#7: DNA chain Unassigned Nucleic Acid


Mass: 1207.870 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 3 types, 3 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-D3T / 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE


Type: DNA OH 3 prime terminus / Mass: 466.169 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O13P3

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Details

Has ligand of interestN
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
IDNameTypeDetailsEntity IDParent-IDSource
1Human LINE-1 ORF2p target-primed reverse transcription complex with EN domain resolvedCOMPLEXThe human LINE-1 retrotransposon mobilizes using the encoded ORF2p protein by a mechanism called target-primed reverse transcription.#1-#70RECOMBINANT
2ORF2pCOMPLEXLINE-1 ORF2p protein#11RECOMBINANT
3Target DNACOMPLEXTarget DNA idealized from a de novo LINE-1 insertion into the FVIII gene#2-#3, #5-#61RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.268 MDaNO
210.206 MDaNO
310.052 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Trichoplusia ni (cabbage looper)7111High 5
32Trichoplusia ni (cabbage looper)7111High 5
43Trichoplusia ni (cabbage looper)7111High 5
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2500 mMpotassium acetateCH3CO2K1
31.5 mMmagnesium acetateMg(CH3COO)21
410 uMzinc acetateZn(CH3COO)21
51 mMDTTC4H10O2S21
60.5 mMPMSFC7H7FO2S1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 22000 nm / Nominal defocus min: 8000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.82 sec. / Electron dose: 60 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1RELION5particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9.8.92model fitting
8ISOLDE1.8model fitting
9UCSF ChimeraX1.8model fitting
11PHENIX1.21.1-5286model refinement
12Servalcat0.4.72model refinement
13RELION5initial Euler assignment
14RELION5final Euler assignment
15RELION5classification
16RELION53D reconstruction
Image processingDetails: All images were processed using RELION5.0 and CryoSPARC 4.5.3
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4568277
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121941 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingChain residue range: 1-1275
Details: Initial model was an alphaFold 2 prediction and was rebuilt into the density.
Source name: AlphaFold / Type: in silico model
RefinementResolution: 3.1→154.71 Å / Cor.coef. Fo:Fc: 0.893 / SU B: 16.343 / SU ML: 0.277 / ESU R: 0.34
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.31862 --
obs0.31862 151900 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 142.267 Å2
Refinement stepCycle: 1 / Total: 12203
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01212649
ELECTRON MICROSCOPYr_bond_other_d0.0010.01611390
ELECTRON MICROSCOPYr_angle_refined_deg1.4671.83117423
ELECTRON MICROSCOPYr_angle_other_deg0.6281.77926416
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.475.3211678
ELECTRON MICROSCOPYr_dihedral_angle_2_deg5.486561
ELECTRON MICROSCOPYr_dihedral_angle_3_deg10.931102113
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.130.2081995
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0213181
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022689
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it9.31613.4475050
ELECTRON MICROSCOPYr_mcbond_other9.31113.4465049
ELECTRON MICROSCOPYr_mcangle_it15.18924.2356309
ELECTRON MICROSCOPYr_mcangle_other15.1924.2356310
ELECTRON MICROSCOPYr_scbond_it10.26314.8147599
ELECTRON MICROSCOPYr_scbond_other10.26214.8147599
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other17.47826.64811115
ELECTRON MICROSCOPYr_long_range_B_refined26.815202.4565723
ELECTRON MICROSCOPYr_long_range_B_other26.815202.4565724
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.1→3.181 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.862 11223 -
obs--100 %

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