[English] 日本語
Yorodumi
- EMDB-40858: Structure of LINE-1 ORF2p with template:primer hybrid -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-40858
TitleStructure of LINE-1 ORF2p with template:primer hybrid
Map dataMain map of LINE-1 ORF2p post-processed with deepEMhancer
Sample
  • Complex: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
  • DNA: DNA primer
  • RNA: RNA template
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsreverse 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 domain / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
LINE-1 retrotransposable element ORF2 protein
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
Authorsvan Eeuwen T / Taylor MS / Rout MP
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_40858.png

Downloads & links

-
Map

FileDownload / File: emd_40858.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map of LINE-1 ORF2p post-processed with deepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 240 pix.
= 206.4 Å		0.86 Å/pix.
x 240 pix.
= 206.4 Å		0.86 Å/pix.
x 240 pix.
= 206.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.281
Minimum - Maximum-0.0017021794 - 1.9721907
Average (Standard dev.)0.0018094959 (±0.03214006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 206.40001 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Mask #1

Fileemd_40858_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Additional map: Main map of LINE-1 ORF2p post-processed with global...

Fileemd_40858_additional_1.map
AnnotationMain map of LINE-1 ORF2p post-processed with global B factor sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #2

Fileemd_40858_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_40858_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Long Interspersed Nuclear Element (LINE)-1 ORF2 protein

EntireName: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
Components
  • Complex: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
  • DNA: DNA primer
  • RNA: RNA template
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

-
Supramolecule #1: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein

SupramoleculeName: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

-
Macromolecule #1: DNA primer

MacromoleculeName: DNA primer / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.975611 KDa
SequenceString:
(DG)(DC)(DG)(DA)(DA)(DA)(DA)(DT)(DT)(DT) (DC)(DG)(DC)

-
Macromolecule #2: RNA template

MacromoleculeName: RNA template / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.444269 KDa
SequenceString:
GUGAGCGAAA UUUUCGC

-
Macromolecule #3: LINE-1 retrotransposable element ORF2 protein

MacromoleculeName: LINE-1 retrotransposable element ORF2 protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 149.252344 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSILDRSTRQ K VNKDTQEL ...String:
MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKI KRDKEGHYIM VKGSIQQEEL TILNIYAPNT GAPRFIKQVL SDLQRDLDSH TLIMGDFNTP LSILDRSTRQ K VNKDTQEL NSALHQTDLI DIYRTLHPKS TEYTFFSAPH HTYSKIDHIV GSKALLSKCK RTEIITNYLS DHSAIKLELR IK NLTQSRS TTWKLNNLLL NDYWVHNEMK AEIKMFFETN ENKDTTYQNL WDAFKAVCRG KFIALNAYKR KQERSKIDTL TSQ LKELEK QEQTHSKASR RQEITKIRAE LKEIETQKTL QKINESRSWF FERINKIDRP LARLIKKKRE KNQIDTIKND KGDI TTDPT EIQTTIREYY KHLYANKLEN LEEMDTFLDT YTLPRLNQEE VESLNRPITG SEIVAIINSL PTKKSPGPDG FTAEF YQRY KEELVPFLLK LFQSIEKEGI LPNSFYEASI ILIPKPGRDT TKKENFRPIS LMNIDAKILN KILANRIQQH IKKLIH HDQ VGFIPGMQGW FNIRKSINVI QHINRAKDKN HVIISIDAEK AFDKIQQPFM LKTLNKLGID GMYLKIIRAI YDKPTAN II LNGQKLEAFP LKTGTRQGCP LSPLLFNIVL EVLARAIRQE KEIKGIQLGK EEVKLSLFAD DMIVYLENPI VSAQNLLK L ISNFSKVSGY KINVQKSQAF LYNNNRQTES QIMGELPFTI ASKRIKYLGI QLTRDVKDLF KENYKPLLKE IKEDTNKWK NIPCSWVGRI NIVKMAILPK VIYRFNAIPI KLPMTFFTEL EKTTLKFIWN QKRARIAKSI LSQKNKAGGI TLPDFKLYYK ATVTKTAWY WYQNRDIDQW NRTEPSEIMP HIYNYLIFDK PEKNKQWGKD SLLNKWCWEN WLAICRKLKL DPFLTPYTKI N SRWIKDLN VKPKTIKTLE ENLGITIQDI GVGKDFMSKT PKAMATKDKI DKWDLIKLKS FCTAKETTIR VNRQPTTWEK IF ATYSSDK GLISRIYNEL KQIYKKKTNN PIKKWAKDMN RHFSKEDIYA AKKHMKKCSS SLAIREMQIK TTMRYHLTPV RMA IIKKSG NNRCWRGCGE IGTLVHCWWD CKLVQPLWKS VWRFLRDLEL EIPFDPAIPL LGIYPKDYKS CCYKDTCTRM FIAA LFTIA KTWNQPNCPT MIDWIKKMWH IYTMEYYAAI KNDEFISFVG TWMKLETIIL SKLSQEQKTK HRIFSLIGGN

UniProtKB: LINE-1 retrotransposable element ORF2 protein

-
Macromolecule #4: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

-
Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
2.0 mMMg(CH3COO)2magnesium acetate
2.0 mMC4H10O2S2DTT

Details: 20mM HEPES pH 7.6, 150mM NaCl, 2mM MgOAc, 2mM DTT, 2mM dTTP
GridPretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: Pelco easy glow
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC / Details: Manually blotted from the back.
DetailsSample was monodisperse though unstable

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 11270 / Average electron dose: 54.0 e/Å2
Details: Super-resolution images collected in dose-fractionation mode over 54 frames with a dose per frame of 1.08 e/A2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

DetailsMovies were binned during motion and gain correction
Particle selectionNumber selected: 1074466
Details: Particles were template picked using 2D class average references
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model
Details: Ab initio model was generated in cryoSPARC from the data
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 430198
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: cryoSPARC (ver. 3.3.1), RELION (ver. 3.1))
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

-
Atomic model buiding 1

Initial modelPDB ID:

8c8j


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 109
Output model

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

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more