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- EMDB-40856: Single particle reconstruction of the human LINE-1 ORF2p without ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40856
TitleSingle particle reconstruction of the human LINE-1 ORF2p without substrate (apo)
Map dataMain map of apo ORF2p core
Sample
  • Complex: Long Interspersed Nuclear Element (LINE)-1 ORF2 protein
    • Protein or peptide: LINE-1 retrotransposable element ORF2 protein
Keywordsreverse transcriptase / LINE-1 / RNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.06 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 23, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40856.png

Downloads & links

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Map

FileDownload / File: emd_40856.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map of apo ORF2p core
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 320 pix.
= 208. Å		0.65 Å/pix.
x 320 pix.
= 208. Å		0.65 Å/pix.
x 320 pix.
= 208. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.104
Minimum - Maximum-0.20860232 - 0.4670853
Average (Standard dev.)0.0014246075 (±0.017033702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40856_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 4.5 angstrom lowpass filtered map

Fileemd_40856_additional_1.map
Annotation4.5 angstrom lowpass filtered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_40856_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_40856_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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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

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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: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: LINE-1 retrotransposable element ORF2 protein

MacromoleculeName: LINE-1 retrotransposable element ORF2 protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKIK RDKEGHYIMV KGSIQQEELT ILNIYAPNTG APRFIKQVLS DLQRDLDSHT LIMGDFNTPL SILDRSTRQK VNKDTQELNS ...String:
MTGSNSHITI LTLNVNGLNS PIKRHRLASW IKSQDPSVCC IQETHLTCRD THRLKIKGWR KIYQANGKQK KAGVAILVSD KTDFKPTKIK RDKEGHYIMV KGSIQQEELT ILNIYAPNTG APRFIKQVLS DLQRDLDSHT LIMGDFNTPL SILDRSTRQK VNKDTQELNS ALHQTDLIDI YRTLHPKSTE YTFFSAPHHT YSKIDHIVGS KALLSKCKRT EIITNYLSDH SAIKLELRIK NLTQSRSTTW KLNNLLLNDY WVHNEMKAEI KMFFETNENK DTTYQNLWDA FKAVCRGKFI ALNAYKRKQE RSKIDTLTSQ LKELEKQEQT HSKASRRQEI TKIRAELKEI ETQKTLQKIN ESRSWFFERI NKIDRPLARL IKKKREKNQI DTIKNDKGDI TTDPTEIQTT IREYYKHLYA NKLENLEEMD TFLDTYTLPR LNQEEVESLN RPITGSEIVA IINSLPTKKS PGPDGFTAEF YQRYKEELVP FLLKLFQSIE KEGILPNSFY EASIILIPKP GRDTTKKENF RPISLMNIDA KILNKILANR IQQHIKKLIH HDQVGFIPGM QGWFNIRKSI NVIQHINRAK DKNHVIISID AEKAFDKIQQ PFMLKTLNKL GIDGMYLKII RAIYDKPTAN IILNGQKLEA FPLKTGTRQG CPLSPLLFNI VLEVLARAIR QEKEIKGIQL GKEEVKLSLF ADDMIVYLEN PIVSAQNLLK LISNFSKVSG YKINVQKSQA FLYNNNRQTE SQIMGELPFT IASKRIKYLG IQLTRDVKDL FKENYKPLLK EIKEDTNKWK NIPCSWVGRI NIVKMAILPK VIYRFNAIPI KLPMTFFTEL EKTTLKFIWN QKRARIAKSI LSQKNKAGGI TLPDFKLYYK ATVTKTAWYW YQNRDIDQWN RTEPSEIMPH IYNYLIFDKP EKNKQWGKDS LLNKWCWENW LAICRKLKLD PFLTPYTKIN SRWIKDLNVK PKTIKTLEEN LGITIQDIGV GKDFMSKTPK AMATKDKIDK WDLIKLKSFC TAKETTIRVN RQPTTWEKIF ATYSSDKGLI SRIYNELKQI YKKKTNNPIK KWAKDMNRHF SKEDIYAAKK HMKKCSSSLA IREMQIKTTM RYHLTPVRMA IIKKSGNNRC WRGCGEIGTL VHCWWDCKLV QPLWKSVWRF LRDLELEIPF DPAIPLLGIY PKDYKSCCYK DTCTRMFIAA LFTIAKTWNQ PNCPTMIDWI KKMWHIYTME YYAAIKNDEF ISFVGTWMKL ETIILSKLSQ EQKTKHRIFS LIGGN

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC / Details: Manually blotted from the back.
DetailsSample was monodisperse though unstable

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6411 / Average electron dose: 51.0 e/Å2
Details: Super-resolution images collected in dose-fractionation mode over 38 frames wiht a dose per frame of 1.32 e/A2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies were binned during motion and gain correction
Particle selectionNumber selected: 1081711
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) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 104980
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)

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